Experimental Research Of Influence Of Pentoxifylline On Human Sperm Function In Vitro

Objective:To research the effect of Pentoxifylline(PF)of different concentrations on human sperm viability,motility rate,motility function,acrosome reaction(AR),plasma membrane functional integrity and intracellular calcium concentration in vitro.To investigate the mechanism of PF provocate AR.Methods:Sperm were aseptically obtained by masturbation and prepared by swim-up technique to produce a spermatozoa solution of high motility from 30 healthy fertile men.Part one,the spermatozoa solution was incubated with the different concentrations of PF(4mM, 8mM,16mM)at 37℃in vitro.Measurements were carried out at 30 and 60 min in 10 specimens,including sperm motility,sperm viability and motility function,while establishing normal control group.Part two,the sperm of 60-minute first part of group semen were carried out the hypo-osmotic swelling(HOS)test,while establishing normal control group.Part three, the spermatozoa solution which were treated by swim-up technique were incubated with the different concentrations of PF(4mM,8mM,16mM)at 37℃in vitro.The FITC-PSA was used to stain the capacitated spermatozoa that have be incubated for 60 min in vitro.Acrosome reaction induced by PF was measured by means of a flowcytometer,while establishing normal control group.Part four,the spermatozoa solution which were treated by swim-up technique was incubated for 30 min with the different concentrations of PF(4mM,8mM,16mM)at 37℃in vitro.Intracellular calcium concentration was measured utilizing the fluorescent probe Fura-3/AM,while establishing normal control group.The fluorescentin tensity of intracellular calcium was measured by RF-450 spectrofluoremeter.Results:Part one,compared with the control group,4mmol/L group,8mmol/L group,16mmol/L group's viability and motility rate have significantly different(P＜0.05),and VCL,VSL,VAP,MAD,ALH,WOB have significantly different(P＜0.05).But,BCF,LIN,STR have not significantly different(P＞0.05),compared with the control group.This indicated sperm viability,motility rate,motility function can maintain high activity trend with increasing PF' concentration and incubated time.Part two,the percentage of g type sperm and the total tail swelling rate were not significantly different(P＞0.05)between control group,4mmol/L group, 8mmol/L group,and 16mmol/L group.This showed PF have no effect on the sperm membrane integrity.Part three,compared with the 16mmol/L group,control group,4mmol/L group, 8mmol/L group,the fluorescent marker values were lower.The fluorescent marker values have significantly different(P＜0.05).There were not significantly different(P＞0.05)between control group,4mmol/L group and 8mmol/L group.This shows that PF of 16mmol/L group can induce AR.Part four,the intracellular calcium fluorescent marker of 16 mmol/L group was significantly higher than the control group and other experimental group.The difference was significant(P＜0.05).Control group,4mmol/L group,8mmol/L group of three experimental groups were higher margin sperm cells calcium fluorescence.However,no significant difference.This shows the mechanism of PF induce AR have relation with intracellular calcium concentration.Conclusions:Pentoxifylline of different concentrations can improve human sperm viability, motility rate and motility function,but it can not modify plasma membrane functional integrity. Pentoxifylline of high concentration can induce acrosome reaction and promote intracellular calcium influx in vitro.The results reveal some internal reasons that PF may improve sperm fertility ability and provide us some for the development of sperm processing technology practical experience.The results also provide us a theoretical basis and theoretical references for the development of artificial fertilization and fertilization technology in vitro.