33 12 Vol.33 No.12 2013 12 Dec. 2013 Reproduction & Contraception doi: 10.7669/j.issn.0253-357X.2013.12.0827 E-mail: randc_journal@163.com 1 2 1 1 1 1 (1. 518020) (2. 518102) : () : 31 2 ; 2 : 2 h (TP) ΔTP( 2 h TP TP ) (AR) ΔAR( AR AR ) (P<0.001) : 1.55 (95%CI=1.04~2.81) 1.81 (95%CI=0.89~6.11) 3.35 (95%CI=1.04~10.32) 1.37 (95%CI=0.96~2.71) 1.88 (95%CI=1.09~4.71); AR DNA (P<0.001) 68%(95%CI=19%~102%) 47%(95%CI=2%~103%) 21%(95%CI=0%~70%); 2 (P>0.05) : - : ; (); ; : Q492.4 R698 +.2 : A : 0253-357X(2013)12-0827-08 [56] IVF () [1] [2-4] 2 [5] : 201102173 : ; Tel/Fax: +86-755-25533000*2085; E-mail: liuyustudio@163.com ( - ) - [7] 2 ( : Sperm Slow) ( : PICSI) - ICSI [89] - ICSI IVF 827
(IUI) ( ); - IVF IUI 1 1.1 : >15 10 6 /ml >32% 3% WHO [10] 2 1.2 1.2.1 80% 40% (PureSperm 40/80) (PureSperm Wash) Nidacon : (Juvederm 30 LEA Derm) ( : 200910105434.7) 1 30 25ml/cm 2 Petri (BD Falcon 1006) γ 1.2.2 WLJY-9000 (CASA) 2123-2 CO2 Shell Lab DMIL Leica 1.2.3 (ARIC) DNA (TP) 1.3 1.3.1 3 - : 1 ml Petri 10 min ( 1); : 5~10 ml 2 ; : 2~5 ml 2 200 g 8 min PureSperm 40/80 2 1.3.2 : CASA ; : WHO [10] Diff-Quik (TZI) (SDI) TZI=( + + )/ : hyaluronic acid coated at the surface of solid state : hyaluronic acid receptor 1 Figure 1 The basic technique principle of spermatozoa selected by solid state hyaluronic acid method 828
SDI=( + + )/ ; TP: 1 10 6 /ml 2 37 5%CO2 2 h( ) ( ); -FITC 4 ; TP ( ) ΔTP(%) TP(%) TP(%); ARIC: 1 10 6 /ml 2 37 5%CO2 3 h Ca 2+ A23187 15 min( ) A23187 15 min( ) -FITC 4 (AR) ; 450~490 nm AR ( ) ΔAR(%) AR(%) AR(%); : 400 ( ) (%)= / 100%; DNA : (SCD) (400 ) 500 DNA DNA : 1/3 1.4 SPSS15.0 t 2 P<0.05 / 2 2.1 2 31 2 (P>0.05) (P<0.001) TZI SDI (P> 0.05)( 1) 1.55 (95%CI=1.04~2.81)( 2A) 2.2 2 2 DNA ( 3A 3B) DNA (P< 0.001)( 1) DNA 47%(95%CI=2%~103%) 21%(95%CI=0%~70%) ( 2B) 2.3 2 2 2 h TP ( 3C) TP ΔTP (P<0.001) 2 TP (P>0.05)( 1) 2 h TP ΔTP 2 h TP 1.81 (95%CI=0.89~6.11) TP (ΔAR) 3.35 (95%CI=1.04~10.32) ( 2C) AR ΔAR AR (P<0.001)( 1) 2 Ca 2+ AR ( 3D) 3 h AR 68%(95%CI=19%~102%); AR ΔAR 1.37 (95%CI=0.96~2.71) 1.88 (95%CI=1.09~4.71)( 2D) 829
1 2 (x s) Table 1 Comparison of the qualities of selected sperm fractions between the two methods Index Solid state hyaluronic acid method Gradient-density centrifugation method Progressive motility (%) 56.47 12.94 58.34 12.79 Sperm motility (%) 78.53 7.80 81.87 10.40 Normal morphology (%) 20.35 9.02 * 14.21 6.26 TZI 1.15 0.19 1.21 0.11 SDI 0.98 0.19 1.04 0.10 Immature sperm nucleoprotein (%) 13.63 10.71 * 32.14 14.59 DNA DNA fragmentation (%) 0.53 0.72 * 2.60 2.39 TP (37 2 h) TP TP after capacitated (%) 27.4 16.86 * 18.95 11.84 TP TP without capacitation (%) 11.38 10.42 11.14 8.82 ΔTP 16.02 11.76 * 7.81 6.60 ARIC (37 3 h) AR Induced AR (+A23187) (%) 63.61 19.01 * 51.18 19.92 AR Spontaneous AR (%) 8.75 4.99 * 14.30 7.50 ΔAR (%) 55.05 18.87 * 36.88 20.86 *: P<0.001 compared with gradient-density centrifugation method A: normal sperm morphology B: sperm immaturity C: TP D: ARIC 2 - (y=1 ) Figure 2 The scatter diagrams and logarithmic trendlines of relative test results of selected sperm by solid state hyaluronic acid technique (y=1 is a base line of the analysis results of separated sperm by gradient-density centrifugation method) 830
1: solid state hyaluronic acid method 2: gradient-density centrifugation method A: aniline blue staining of sperm nucleoprotein ( : immature nucleoprotein) B: DNA sperm DNA fragmentation ( : DNA DNA fragmentation) C: 2 h TP TP after capacitated for 2 h ( : localization of protein tyrosine phosphorylation on sperm tail) D: 3 h AR A23187-induced AR after capacitated for 3 h ( : acrosome reaction) 3 2 Figure 3 Comparison of the results of stained sperm between the two methods 831
3 [10] IUI IVF ARIC ; ROC ARIC IUI 85.3% 85.5% [1819] ( AR) [20] AR - IVF - (P>0.05) AR ΔAR - AR ΔAR AR - [11] PICSI ; [9] (MSOME) [12] (8 400 ) MSOME Ca 2+ HCO3 camp [13-16] [17] 2 h TP - (r=0.771 P<0.001) TP - TP ( TP) 2 h TP ΔTP - ARIC Ca 2+ A23187 Ca 2+ [21] ICSI (37.4% vs 30.7%) (50.8% vs 37.9%) - ( Sperm Slow PICSI) ICSI ICSI - DNA DNA [6922] - [2324] [25] ; DNA [26] 17 4~6 - (r= 0.682 P<0.001) [27] Worrilow [21] - 65% PICSI ICSI 832
( ) (0%~3.3% vs 15.1%~18.5% P<0.05) - [24] [21] - : ; IUI IVF [1] Drahorád J Tesarík J Cechová D et al. Proteins and glycosaminoglycans in the intercellular matrix of the human cumulusoophorous and their effect on conversion of proacrosin to acrosin. J Reprod Fertil1991 93(2): 253-62. [2] Kornovski BS McCoshen J Kredentser J et al. The regulation of sperm motility by a novel hyaluronan receptor. Fertil Steril 1994 61(5):935-40. [3] Ranganathan S Ganguly AK Datta K. Evidence for presence of hyaluronan binding protein on spermatozoa and its possible involvement in sperm function. Mol Reprod 1994 38 (1):69-76. [4] Lin Y Mahan K Lathrop WF et al. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer. J Cell Biol 1994 125(5): 1157-63. [5] Huszar G Jakab A Sakkas D et al. Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects. Reprod Biomed Online 2007 14(5):650-63. [6] Huszar G Ozenci CC Cayli S et al. Hyaluronic acid binding by human sperm indicates cellular maturity viability and unreacted acrosomal status. Fertil Steril 2003 70(3):1616-24. [7] Parmegiani L Cognigni GE Bernardi S et al. Comparison of two ready-to-use systems designed for sperm-hyaluronic acid binding selection before intracytoplasmic sperm injection: PICSI vs Sperm Slow: a prospective randomized trial. Fertil Steril 2012 98(3):632-7. [8] Parmegiani L Cognigni GE Ciampaglia W et al. Efficiency of hyaluronic acid () sperm selection. J Assist Reprod Genet 2010 27(1):13-6. [9] Parmegiani L Cognigni GE Bernardi S et al. Physiologic ICSI : hyaluronic acid () favors selection of spermatozoa without DNA fragmentation and with normal nucleus resulting in improvement of embryo quality. Fertil Steril 2010 93(2):598-604. [10] WHO. Laboratory manual for the examination and processing of human semen. 5 ed. Geneva: WHO Press 2010. [11] Prinosilova P Kruger T Sati L et al. Selectivity of hyaluronic acid binding for spermatozoa with normal tygerberg strict morphology. Reprod Biomed Online 2009 18(2):177-83. [12] Petersen CG Massaro FC Mauri AL et al. Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification. Reprod Biol Endocrinol 2010 8(12):149. [13] Leclerc P de Lamirande E Gagnon C. Cyclic adenosine 3'5'- monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996 55(3):684-92. [14] Visconti PE Kopf GS. Regulation of protein phosphorylation during sperm capacitation. Biol Reprod 1998 59(1):1-6. [15] Nassar A Mathony M Morshedi M et al. Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility. Fertil Steril 1999 71(5):919-23. [16] Urner F Sakkas D. Protein phosphorylation in mammalian spermatozoa. Reproduction 2003 125(1):17-26. [17] Liu DY Clarke GN Baker HW. Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm-zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction. Hum Reprod 2006 21(4):1002-8. [18] Makkar G Ng EH Yeung WS et al. The significance of the ionophore-challenged acrosome reaction in the prediction of successful outcome of controlled ovarian stimulation and intrauterine insemination. Hum Reprod 2003 18(3):534-9. [19] Katsuki T Hara T Ueda K et al. Prediction of outcomes of assisted reproduction treatment using the calcium ionophoreinduced acrosome reaction. Hum Reprod 2005 20(2):469-75. [20] Liu DY Garrett C Baker HWG. Acrosome reacted sperm in medium do not bind to the zona pellucida of human oocytes. Int J Androl 2006 29(4):475-81. [21] Worrilow KC Eid S Woodhouse D et al. Use of hyaluronan in the selection of sperm for intracytoplasmic sperm injection (ICSI): significant improvement in clinical outcomes-- 833
multicenter double-blinded and randomized controlled trial. Hum Reprod 2013 28(2):306-14. [22] Yagci A Murk W Stronk J et al. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study. J Androl 2010 31(6):566-72. [23].. 2010 24(7): 21-5. [24].. 2011 31(8):532-7. [25] Zini A Borman JM Belzile E et al. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic reviewand meta-analysis. Hum Reprod 2008 23(12):2663-8. [26] Jakab A Sakkas D Delpiano E et al. Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies. Fertil Steril 2005 84(6):1665-73. [27] Mokánszki A Molnár Z Ujfalusi A et al. Correlation study between sperm concentration hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients. Reprod Biomed Online 2012 25(6):620-6. (2013 6 13 ) Pilot Evaluation of Sperm Selection Procedure Based on Solid State Hyaluronic Acid Binding Ability on Improving Sperm Qualities Yu LIU 1 Jing ZNG 2 Jin-e CHENG 1 Xiao-lan CHEN 1 Wen-yuan WU 1 Dai-ni ZHENG 1 (1. Laboratory of Reproduction Medicine Shenzhen People s Hospital Shenzhen 518020) (2. Shenzhen Baoan Chronic Diseases Prevent and Cure Hospital Shenzhen 518102) ABSTRACT Objective: To explore the advantages of solid state hyaluronic acid () selection procedure for high throughput sperm separation compared with traditional gradient centrifugation (GC) method in the aspect of separated sperm qualities. Methods: Totally 31 fresh semen samples were collected. Every sample was divided into 2 parts. One part was applied to the bottom surface of Petri dishes which pre-coated by immobilized. The unbound sperm were rinsed gently and the -bound sperm were removed and collected from dish surface. Another part of semen was separated synchronically using GC method. The qualities of separated sperm were compared between the two groups and calculation the extent of function indexes were promoted of selected sperm were compared by solid state technique. Results: In the -bound sperm fractions compared with GC-selected sperm fractions the proportions of normal morphology protein tyrosine phosphorylation (TP) after capacitated for 2 h (2 h TP) TP score ( 2 h TP minus sperm TP without capacitation ) A23187-induced acrosome reaction (AR) and AR score ( induced AR minus spontaneous AR ) were higher (P<0.001) and were increased to 1.55-fold (1.04 2.81) 1.81-fold (0.89 6.11) 3.35-fold (1.04 10.32) 1.37-fold (0.96 2.71) and 1.88-fold (1.09 4.71) respectively. Conversely the levels of spontaneous AR immature nucleoproteinand DNA fragment were lower by solid state technique than by GC method (P<0.001) and were reduced to 68% (19% 102%) 47% (2% 103%) and 21% (0% 70%) respectively. However there was no significant difference in percentages of sperm progressive motility and total motility between the two methods (P>0.05). Conclusion: The solid state sperm selection technique which is based on a specific binding capacity between sperm receptors for zona pellucida and exogenous will likely increase the ability of spermatozoan fertilization and reduce immaturity and will be promising in improving the outcome of assisted reproduction. Key words: solid state; hyaluronic acid (); sperm preparation; sperm function 834