The Pilot Study Of Sperm Apoptosis's Relationship With Human Semen Parameters And Its Influence Factors

BACKGROUNDInfertility is approximately 10%-20% couples reproductive age, andmale factors are about 40%. The etiological factors of male infertility arecomplicated. At present, majority person think highly of male sterility byhormone diversity, immune response, reproductive system infection ormalformation and so on. It was ignored individual inheritance factor andcontinue factor, resulting in infertility question by abnormal of spermapoptosis. Recently document indicated, with the development of modernsociety industrialization,harm secondary effect and genetic fators lead tosperm quantity and quality were generally decreased tendency, such asradiation, medicine and toxin and so on. This is apoptotic characteristic withsperm DNA fragment and membrane phosphatidylserine (PS) translocationon male infertility.At present, clinically detection sperm functional methodssill impossible identity those which had already occurred apoptotic sperm.However, investigation had already made clear that the extremely closerelationship between sperm apoptosis and male infertility.In recent years, Intracytoplasmic injection ( ICSI ) technique hadbecome one of assisted reproductive technique by treated male infertility.When ICSI will those valbility low, abnormal of acrosome reaction or spermof sperm-egg abnormal fusion injected ovum, meanwhile, may also injectsperm of process apoptosis characteristic.Oocyte into impairedgenome,which will result in abnormal of embryonic development orchromosome of some influence male infertile abnormal or gene offspring,new born of ICSI off spring potentially reproducing its father of steriletragedy after adolescence. Therefore, the study relationship between spermapoptosis and male infertility will contribute to infertility patients etiologicaldiscriminate and treating of diagnose.OBJECTIVESSperm apoptosis detection could be regarded as a important markerfor evaluation of male fertility. Our study demonstrated important role ofsperm apoptosis analyse in male infertility the relationship between spermapoptosis and semen routine main parameters (motion, viability, liquefiedduration, sperm concentration and leukocyte concentration factors and soon).our results will reveal sperm apoptosis induced factors which providedexperiment based on and try to explain the mechanism of these factors onmale infertility. For sperm apoptosis established preliminary investigationrationale by the apoptosis of the human spermatogenic cells ultrastructuralorganization analysis. The study also showed that sperm separation willimprove the outcome of assistant reproduction technology, partly because itcan select influenced sperm apoptosis.METHODSAll semem samples came from Clinical Hospital of Jilin University.From 7 month 2005 to 2 month 2006, We have collected seminal specimensof male infertility, and classified these specimens according to abnormalfactors.Their age ranged from 25 to 37 years.All the patients suffered fromprimary infertility of more than one years duration and no injury, geneticdesease family history and sexual examination case history. Healthexamination detected no abnomal of existent obviously testicle, epididymisand deferens. All patients had a normal physical examination. All thesesamples were from 24 fertility persons. Their age ranged from 22 to 36years. Basic semen parameters, including sperm concentration, motility,viability and morphology, were assessed, which had reproduction andhealth male, according to the World Health Organization (WHO) guidelines.All samples used in this study were normal according to the WHO criteria(World Health Organization,1999).In our experiments the sperm apoptosiswere analyzed by Wright-Giemsa staining and the semen parameters weredetermined by computer assisted sperm analysis (CASA). Detection ofmembrane phosphatidylserine (PS) translocation were used by annexin VCy3.18 (Apoptosis Detection Kit,Annexin V-Cy3;Sigma). Generation ofreactive oxygen species (ROS) was measured by a chemiluminescenceassay using luminal. The apoptosis of the human spermatogenic cellsultrastructure of male infertility by electron microscopy. Sperm wereseparated using discontinuous Percoll gradient separation .RESULTS①The percentages of sperm apoptosis of infertility group wereobviously higher than that of fertility group(P<0.01). ②The percentages ofsperm apoptosis of groups of oligozoospermia, asthenozoospermia andteratozoospermia were significantly higher than fertility group (P<0.01,P<0.05),respectively. ③There are negative significantly correlationshipsbetween sperm concentration, motility rate of sperms, a,b grade sperms ,semen liquefied duration and percentages of sperm apoptosis(r=-0.35,-0.52,-0.32,-0.41, P<0.05 ,P<0.01), respectively. Semen leukocyteconcentration were positive correlated significantly with percentages ofsperm apoptosis (r =0.31,P<0.05). Motion parameters of sperms: VCL,VAP, VSL are negative correlated significantly with percentages of spermapoptosis(r=-0.34,-0.34 ,P<0.05). There are no correlationships betweenVCL, MAD, ALH, BCF,LIN, WOB, STR and percentages of spermapoptosis.Our results exhibited the percentage of sperm apoptosis in a,bgrade motile sperm abnormal group(Md=25%,QR=20%) was respectivelyhigher than corresponding control group(Md=9%,QR=16%) (P<0.05). Agrade, b grade, c grade, a,b grade motile sperm a,b,c,grade motile spermwere negative correlated significantly with percentages of sperm apoptosis.D grade were positive correlated significantly with percentages of spermapoptosis (P<0.01).There were significantly between percentages of spermapoptosis of viability abnormal groups(Md=25%,QR=20%)and thatnormal groups(Md=9%,QR=23%) (P<0.05).The percentages of spermapoptosis of sperm concentration normal group were obviously lower thanthat of abnormal group (P <0.05). The percent of sperm apoptosis was notsignificantly different between the normal group(Md=14%,QR=25%) andabnormal group(Md=26%, QR=40%) of semen volume(P >0.05).Therewere significantly between percentages of sperm apoptosis of semenleukocyte concentration abnormal groups(Md=30%,QR=33%) and thatnormal groups(Md=9%,QR=26%) (P <0.05). The percent of spermapoptosis was not significantly different between the normal group andabnormal group of sperm morphology(P >0.05).④The percentages ofsperm apoptosis of seminal plasma zinc concentration abnormal groupswere obviously higher than that of zinc normal groups (P<0.05).⑤Reactive oxygen species and the percentages of sperm apoptosishave positive significantly correlationships (P <0.05). High motility spermgroups of after percoll selection treated with two different concentrations ofhydrogen peroxide(H2O2) after 30 min, the percentages of sperm apoptosiswas respectively higher than corresponding control group(P<0.001). Lowmotility sperm groups of after percoll selection treated with two differentconcentrations of H2O2 after 30min,the percentages of sperm apoptosis wasrespectively higher than corresponding control group ( P<0.05,P<0.001).Our results exhibited the percentage of sperm apoptosis insmoking group was respectively higher than nonsmokers group( P<0.05).⑥The percentages of sperm apoptosis were significantly decreased aftersperm selection technique(P <0.05).⑦The apoptosis of the humanspermatogenic cells showed the concentration and the increased electronicdensityof the nuclear chromatin.the enlargement and bulginess of the spaceof the nuclear membrane and with the vacuoles formation.Theexpansion,folding,Enveloping with the rosette patterns of the nuclearmembrane.Abnormal mitochondria was appeared that different size,abnormal morphology , inhomogeneous distribution or abnormalarrangement.CONCLUSIONS①Sperm apoptosis detection could be regarded as a importantmarker for evaluation of male fertility. ②Obviously, detection of spermapoptosis in sperm samples may help evaluate sperm quality, which coulddecrease for evaluation of male fertility.③Our study provided evidencesthat semen leukocyte concentration abnormal maybe induce spermapoptosis.④ O ur results showed seminal plasma zinc contents abnormalcould induced sperm apoptosis.⑤ Reactive oxygen species and smokingcould maybe induce sperm apoptosis factors.The data indicated that thehigh dose of H2O2 induced the appearance of phosphatidylserine on thememebranes both in the high and low motility fractions. ⑥The percentagesof sperm apoptosis were significantly decreased after sperm selectiontechnique. ⑦The apoptosis of the human spermatogenic cells showed theconcentration and the increased electronic densityof the nuclearchromatin.the enlargement and bulginess of the space of the nuclearmembrane and with the vacuoles formation,enveopi-ng with the rosettepatterns of the nuclear membrane. Abnormal mitochondria was appearedthat different size, abnormal morphology,inhomogeneous distribution orabnormal arrangement. The apoptosis of the spermatozoa cells and theapermatozoa in an abnormal phenotype in the infertile patients. It isimportant to examine by electron microscopy the spermatogenic cells of thepatients weith infertility in order to detect their ultrastructural changes.