Detection Of IL-23 In Human Seminal Plasma And Relationship With Semen Quality

INTRODUCTIONThe relationship between cytokines and human reproduction represents a growing area of investigation because of their involvement in different aspects of reproductive physiology and fertility regulation.The human semen contains a repertoire of cytokines including transforming growth factor,epidermal growth factor,turnout necrosis factor-α,various interleukines,intefferon-γand certain of their soluble recepters.They are secreted by different parts of the male genital and may exert effects on steroidogenesis,spermatogenesis and sperm function.In human seminal plasma,Inflammatory cytokines are produced by white blood cells,mainly by macrophages,in response to foreign antigens,pathogens and also in chronic inflammation.These cytokines included mainly IL-1,IL-6,IL-8,IL-12,TNF-α,IFN-γ, et al.In-vitro,cytokines(IL-1,IL-6,TNF-α,IFN-γ)affect human sperm motility, increase the production of reactive oxygen species by human spermatozoa and reduce the ova-penetrating ability of spermatozoa.Il-1 and TNF-αinhibite the testosterone production.TNF-αinhibits the sperm acrosin activity and acrosome reaction.Of course,some of these effects are still subject to debate.Interleukin-23(IL-23)is a recently discovered heterodirneric cytokine.It consists of a private cytokine-like p19 subunit and common p40 subunit of IL-12/IL-23 by disulfide bond.It is mainly produced by activated macrophages and dendritic cells(DC),it exerts effects through binding its receptors on macrophages and T cells.IL-23 has been shown to possess multiple biological activities.For example,it can enhance T cells especially CD4~+ T cell proliferation and IFN-γ,IL-12 production from T cells,myeloid antigen-presenting cells(APCs).It can also modulate the costimulatory function of dendritic cells. Additionally,recent studies demonstrated that IL-23 has antiturnor and antimetastatic activity,and it may also be involved in the development of autoimmune diseases and inflammatory diseases.In the transgenic mice study,ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation,runting,infertility,and premature death.To our knowledge,there has no study about relationship between IL-23 and human semen quality.In the present study,sperm parameters and sperm morphology was analyzed according to World Health Organization criteria.On this foundation,the concentrations of IL23,IL-6,IL-8 and TNF-αwere determined in the seminal plasma.Serum FSH,LH,T were determined in the meantime.The correlation between IL-23 concentration with IL-6,IL-8,TNF-αconcentration in seminal plasma, serum concentrations of FSH,LH,T and sperm parameters were analysed.Now the result will be reported as follows.MATERIALS AND METHODSSemen samples57 semen samples were obtained from 57 males:45 unmarried semen donors from Zhejiang human sperm bank and 12 male infertility patients from Zhejiang Institute of Planned Parenthood Research from May 2007 to June 2007.None of the patients was treated with antibiotics before study beginning.The study did not exclud azoospermia and leukocytospermic.The ages of the donors ranged from 20 to 25 years(average, 21.8),and those of the patients were from 26 to 38 years(average,30.1).The genitals, the volume of two testicles and the results of the urine generally checked were normal in all men.57 semen samples were divided into two groups according to semen analysis results. Normal semen quality group(n=35),sperm count≥20×10~6/ml and progressive(a+b)≥40%.Abnormal semen quality group(n=22),sperm count＜20×10~6/ml and/or progressive(a+b)＜40%. Specimens collectionSemen samples were obtained in hospital,ejaculate into sterile container by masturbation after abstinence for 3-5 days,and were examined directly after liquefaction.Semen analysisStandard semen analysis according to World Health Organization criteria(WHO,1999) included determination of volume,PH,count,progressive motility,viability(eosin testing)after liquefaction(at 37℃).Sperm morphology analysisSperm morphology was analyzed according to World Health Organization criteria (WHO,1999).Preparation of seminal plasmaAt 30 min after collection,liquefied semen samples were centrifuged at 3000rpm/min for 20 min.After which,the clear seminal plasma was collected and stored at -20℃until determination of IL-23,IL-6,IL-8 and TNF-αconcentrations.Determination of IL-23,IL-6,IL-8 and TNF-αin seminal plasmaFor detection of IL-23,IL-6,IL-8 and TNF-α.in Seminal plasma,commercial kits were used(shanghai boguang).They were solid phase enzyme-linked immunosorbent assay(ELISA)with quantitative sandwich technique.Determination of FSH,LH and T in serumThe levels of FSH,LH and T were measured by Automatedchemiluminescence system.Statistical analysisData were processed by Statistics Package of Social Science(SPSS)11.5.All values were presented as mean±SD.Statistical analysis was conducted using Welch's t-test. The correlation was analyzed by simple linear regression.The level of significance was set at P＜0.05.RESULTSThe sperm count,sperm progressive motility,sperm motility,sperm viability and normal morphology of the group with normal semen quality was markedly higher comparing with abnormal semen quality group.There was no difference in PH.IL-23 was detected in all semem samples.Its concentration was 8.19±4.60pg/ml (1.44-19.35pg/ml).IL-23 concentration was 6.37±3.58pg/ml in the group with normal semen quality. In the group with abnormal semen quality,IL-23 concentration was markedly higher(11.08±4.63 pg/ml)comparing with the normal group(P＜0.01).IL-23 concentrations were significantly higher in samples with progressive motility＜50% (P＜0.001),IL-23 concentration and progressive motility were significantly negative correlation(r=-0.429,P＜0.01).The concentration of IL-23 and normal morphology rate were also significantly negative correlation(r=-0.266,P＜0.05).There were also significantly positive correlation between IL-23 concentration with IL-6,IL-8,TNF-αconcentration in seminal plasma.There were no marked association with FSH,LH and T in serum.CONCLUSIONSThe human semen contains IL-23.Its concentration was 6.37±3.58pg/ml in the group with normal sperm concentration and progressive motility.IL-23 concentration in the group with abnormal sperm concentration and progressive motility was markedly higher(11.08±4.63pg/ml,P＜0.01).There were significant negative correlation between IL-23 concentration and progressive motility,normal sperm morphology rate. There was also marked positive correlation between IL-23 concentration and IL-6,IL-8,TNF-αconcentrations in seminal plasma.IL-23 concentration was not significantly related to the serum concentrations of FSH,LH,T.