The efficacy of novel centrifugation-free sperm selection (Io-Lix) on sperm parameters and ICSI reproductive outcomes

Published:November 05, 2022DOI:


      Research question

      What is the effect of a novel non-centrifugation method (Io-Lix) of sperm selection on sperm parameters and intracytoplasmic sperm injection (ICSI) reproductive outcomes?


      This pilot study elevated the capacity of the Io-Lix sperm selection protocol to improve sperm parameters (concentration, motility and sperm DNA fragmentation) of the neat ejaculate. Once established, the reproductive outcomes of Io-Lix selected spermatozoa were used for autologous and donor oocyte ICSI programmes and their efficacy compared with those using conventional swim-up.


      Io-Lix sperm selection resulted in lower sperm concentration yield (P < 0.001) and sperm DNA fragmentation (P < 0.001) but higher sperm motility (P < 0.001) when compared with spermatozoa in the neat ejaculate. When compared with swim-up sperm selection the Io-Lix protocol resulted in a 14.7% (P = 0.028) increase in pregnancy rate and 16.3% (P = 0.047) reduction in miscarriages in the autologous ICSI programme. A similar comparison of sperm selection procedures employed for a donor oocyte ICSI programme showed no difference in terms of their respective reproductive outcomes.


      The Io-Lix sperm selection protocol resulted in improved pregnancy rate and reduction in miscarriage when applied to autologous ICSI, which was attributed to a reduction in the proportion of spermatozoa with DNA damage post-selection. A similar finding was not apparent in the donor oocyte programme, which may be associated with the capacity of the donor oocyte to repair sperm DNA post-syngamy.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Reproductive BioMedicine Online
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Ainsworth C.
        • Nixon B.
        • Aitken R.J.
        Development of a novel electrophoretic system for the isolation of human spermatozoa.
        Hum. Reprod. 2005; 20 (Epub 2005 Apr 14. PMID: 15831507): 2261-2270
        • Aitken R.J.
        • Clarkson J.S.
        Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques.
        J. Androl. 1988; 9: 367-376
        • Bareh G.M.
        • Jacoby E.
        • Binkley P.
        • Chang T.C.
        • Schenken R.S.
        • Robinson R.D.
        Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: a prospective study.
        Fertil. Steril. 2015; 105 (329–336.e1)
        • Benchaib M.
        • Lornage J.
        • Mazoyer C.
        • Lejeune H.
        • Salle B.
        • Guerin F.J.
        Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.
        Fertil. Steril. 2007; 87: 93-100
        • Borini A.
        • Tarozzi N.
        • Bizzaro D.
        • Bonu M.A.
        • Fava L.
        • Flamigni C.
        • Coticchio G.
        Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.
        Hum. Reprod. 2006; 21: 2876-2881
        • Carrell D.T.
        • Liu L.
        • Peterson C.M.
        • Jones K.P.
        • Hatasaka H.H.
        • Erickson L.
        • Campbell B.
        Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss.
        Arch. Androl. 2003; 49: 49-55
        • Cuevas-Saiz I.
        • Pons-Gatell M.C.
        • Cuadros-Vargas M.
        • Delgado-Mendive A.
        • Rives-Enedáguila N.
        • Moragas-Solanes M.
        • Carrasco-Canal B.
        • Teruel-López J.
        • Busquets-Bonet A.
        • Hurtado de Mendoza M.V.
        The Embryology Interest Group: updating ASEBIR's morphological scoring system for early embryos, morulae and blastocysts.
        Medicina Reproductiva y Embriología Clínica. 2018; 5: 42-54
        • Dickey R.P.
        • Pyrzak R.
        • Lu P.Y.
        • Taylor S.N.
        • Rye P.H.
        Comparison of the sperm quality necessary for successful intrauterine insemination with World Health Organization threshold values for normal sperm.
        Fertil. Steril. 1999; 71: 684-689
        • Gosálvez J.
        • Caballero P.
        • López-Fernández C.
        • Ortega L.
        • Guijarro J.A.
        • Fernández J.L.
        • Johnston S.D.
        • Nuñez-Calonge R.
        Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?.
        Asian J. Androl. 2013; 15: 812-818
        • Gosálvez J.
        • López-Fernández C.
        • Fernández J.L.
        • Esteves S.C
        • Johnston S.D.
        Unpacking the mysteries of sperm DNA fragmentation: ten frequently asked questions.
        J. Reprod. Biotechnol. Fertil. 2015; 4: 1-16
        • Gosálvez J.
        • López-Fernández C.
        • Fernández J.L.
        • Gouraud A.
        • Holt W.V.
        Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species.
        Mol. Reprod. Dev. 2011; 78: 951-961
        • Gualtieri R.
        • Kalthur G.
        • Barbato V.
        • Longobardi S.
        • Di Rella F.
        • Adiga S.K.
        • Talevi R.
        Sperm oxidative stress during in vitro manipulation and its effects on sperm function and embryo development.
        Antioxidants (Basel). 2021; 10: 1025
        • Iwasaki A.
        • Gagnon C.
        Formation of reactive oxygen species in spermatozoa of infertile patients.
        Fertil. Steril. 1992; 57: 409-416
        • Jayaraman V.
        • Upadhya D.
        • Narayan P.K.
        • Adiga S.K.
        Sperm processing by swim-up and density gradient is effective in elimination of sperm with DNA damage.
        J. Assist. Reprod. Genet. 2012; 29: 557-563
        • Le Lannou D.
        • Blanchard Y.
        Nuclear maturity and morphology of human spermatozoa selected by Percoll gradient centrifugation or by swim-up procedure.
        J. Reprod. Fertil. 1998; 84: 551-556
        • Marchetti F.
        • Essers J.
        • Kanaar R.
        • Wyrobek A.J.
        Disruption of maternal DNA repair increases sperm-derived chromosomal aberrations.
        PNAS. 2007; 104: 17725-17729
        • McQueen D.B.
        • Zhang J.
        • Robins J.C.
        Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis.
        Fertil. Steril. 2019; 112 (54–60.e3)
        • Mortimer D.
        Sperm recovery techniques to maximize fertilizing capacity.
        Reprod. Fertil. Dev. 1994; 6: 25-31
        • Muratori M.
        • Tarozzi N.
        • Cambi M.
        • Boni L.
        • Iorio A.L.
        • Passaro C.
        • Luppino B.
        • Nadalini M.
        • Marchiani S.
        • Tamburrino L.
        • Forti G.
        • Maggi M.
        • Baldi E.
        • Borini A.
        Variation of DNA fragmentation levels during density gradient sperm selection for assisted reproduction techniques: a possible new male predictive parameter of pregnancy?.
        Medicine (Baltimore). 2016; 95: e3624
        • Muratori M.
        • Tarozzi N.
        • Carpentiero F.
        • Danti S.
        • Perrone F.M.
        • Cambi M.
        • Casini A.
        • Azzati C.
        • Boni L.
        • Maggi M.
        • Borini A.
        • Baldi E.
        Sperm selection with density gradient centrifugation and swim up: effect on DNA fragmentation in viable spermatozoa.
        Sci. Rep. 2019; 9: 7492
        • O'Flaherty C.
        Iatrogenic genetic damage of spermatozoa.
        Adv. Exp. Med. Biol. 2014; 791: 117-135
        • Robinson L.
        • Gallos I.D.
        • Conner S.J.
        • Rajkhowa M.
        • Miller D.
        • Lewis S.
        • Kirkman-Brown J.
        • Coomarasamy A.
        The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis.
        Hum. Reprod. 2012; 27: 2908-2917
        • Samuel R.
        • Feng H.
        • Jafek A.
        • Despain D.
        • Jenkins T.
        • Gale B.
        Microfluidic-based sperm sorting & analysis for treatment of male infertility.
        Transl. Androl. Urol. 2018; 7: S336-S347
        • Sánchez-Martín P.
        • Dorado-Silva M.
        • Sánchez-Martín F.
        • González Martínez M.
        • Johnston S.D.
        • Gosálvez J.
        Magnetic cell sorting of semen containing spermatozoa with high DNA fragmentation in ICSI cycles decreases miscarriage rate.
        Reprod. Biomed. Online. 2017; 34: 506-512
        • Santiso R.
        • Tamayo M.
        • Gosálvez J.
        • Johnston S.
        • Mariño A.
        • Fernández C.
        • Losada C
        • Fernández J.L.
        DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide.
        Mutat. Res. 2012; 734: 41-49
        • Santiso R.
        • Tamayo M.
        • Gosálvez J.
        • Meseguer M.
        • Garrido N.
        • Fernández J.L.
        Swim-up procedure selects spermatozoa with longer telomere length.
        Mutat. Res. 2010; 688: 88-90
        • Sharma R.K.
        • Vemulapalli S.
        • Kohn S.
        • Agarwal A.
        Effect of centrifuge speed, refrigeration medium, and sperm washing medium on cryopreserved sperm quality after thawing.
        Arch. Androl. 1997; 39: 33-38
        • Stringer J.M.
        • Winship A.
        • Zerafa N.
        • Wakefield M.
        • Hutt K.
        Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health.
        PNAS. 2020; 117: 11513-11522
        • Tan J.
        • Taskin O.
        • Albert A.
        • Bedaiwy M.A.
        Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: a systematic review and meta-analysis.
        Reprod. Biomed. Online. 2019; 38: 951-960
        • Tvrdá E.
        • Arroyo F.
        • Ďuračka M.
        • López-Fernández C.
        • Gosálvez J.
        Dynamic assessment of human sperm DNA damage II: the effect of sperm concentration adjustment during processing.
        J. Assist. Reprod. Genet. 2019; 36: 799-807
        • van Voorhis B.J.
        • Barnett M.
        • Sparks A.E.
        • Syrop C.H.
        • Rosenthal G.
        • Dawson J.
        Effect of the total motile sperm count on the efficacy and cost-effectiveness of intrauterine insemination and in vitro fertilization.
        Fertil. Steril. 2001; 75: 661-668
        • Wang M.
        • Sun J.
        • Wang L.
        • Gao X.
        • Lu X.
        • Wu Z.
        • Wang Y.
        • Liu K.
        • Tao J.
        • Wu Y.
        Assessment of density gradient centrifugation (DGC) and sperm chromatin dispersion (SCD) measurements in couples with male factor infertility undergoing ICSI.
        J. Assist. Reprod. Genet. 2014; 31: 1655-1663
        • Yifu P.
        • Lei Y.
        • Shaoming L.
        • Yujin G.
        • Xingwang Z.
        Sperm DNA fragmentation index with unexplained recurrent spontaneous abortion: a systematic review and meta-analysis.
        J. Gynecol. Obstet. Hum. Reprod. 2020; 26101740


      Yolanda Cabello is senior clinical embryologist and scientific lead at Overture Life, Madrid. For over 20 years, she has served as director of embryology, andrology and PGT laboratories of various assisted reproduction units. She has been Chief Investigator of five funded European projects, and co-author of 45 peer-reviewed journal articles and seven book chapters.
      Key message
      Io-Lix sperm selection resulted in an improved pregnancy rate when applied to autologous ICSI, which was attributed to a reduced proportion of spermatozoa with DNA damage. A similar finding was not apparent in a donor oocyte ICSI programme associated with the capacity of the oocyte to repair paternal DNA post-syngamy.