Interaction Between Leptin And Epididymitis And Its Effect On Male Fertility

ObjectivesTo explore the interaction between leptin and epididymitis,to clarify whether it has an effect on male fertility,and to clarify the molecular regulatory mechanism of leptin-epididymitis interaction.Materials and Methods1.946 infertile patients from January 2014 to January 2016 in the Department of andrology of our hospital were enrolled as research objects,and divided into four groups: chronic epididymitis group,leptin elevation group,combined with chronic epididymitis and leptin elevation group,and control group(without chronic epididymitis and leptin is normal).General information of patients,leptin hormone and semen parameters,etc.were collected.T-test,chi-square test and covariance analysis(ANCOVA)were used to compare the relationship between different indicators in different groups.2.Establish rat epididymitis animal model,detect the expression of inflammatory factors IL-1beta,TNF-alpha,IL-6 in epididymis tissue and inflammatory animal model;detect the expression of Leptin and its receptor Ob-R in epididymis tissue by immunohistochemistry;observe the difference of sperm quality between model group and control group;screen the differential genes between model group and control group by transcription group sequencing(RNA-seq),Real-time-time-seq.Differential gene expression was verified by PCR and detected by Western blot.3.Isolation of epididymal epithelial cells,treatment with lipopolysaccharide to induce inflammation of epididymal epithelial cells,treatment with leptin and pyrrolidine dithiocarbamate(PDTC),detection of cell apoptosis by CCK,detection of signal pathway genes and expression of inflammatory genes by Real-time PCR,determination of IL-1beta and TNF-alpha secretion by ELISA,and to explore the effects of leptin on epididymitis and the possible signaling pathways involved.Results1.Sperm motility and normal morphology in patients with chronic epididymitis and leptin elevation were lower than those in control group.Sperm density,motility,normal morphology and neutral alpha-glucosidase in patients with chronic epididymitis and leptin elevation were lower than those in patients with chronic epididymitis alone.Sperm motility and neutral alpha-glucosidase also decreased significantly compared with patients with leptin elevation.ANCOVA analysis and correction included the effects of BMI,smoking,infertility types and other confounding factors.The results showed that chronic epididymitis could independently affect the normal sperm morphology rate.Elevated leptin could independently affect sperm density and normal sperm morphology rate,while combined epididymitis and elevated leptin could affect sperm density,sperm motility and normal sperm morphology rate.2.The sperm abnormality rate of the epididymitis model group was significantly higher than that of the control group,the sperm motility was significantly lower than that of the control group,the difference was significant;the sperm number was less than that of the control group,but the results were not statistically significant;the immunohistochemical results showed that the expression of leptin and its receptor in the epididymitis model group was increased;the four down-regulated genes Col28a1 and Cdkl1 were confirmed by high-throughput sequencing and q PCR.Psph,Fads2 and 11 up-regulated genes Cebpb,Cxcr2,Il11,Cxcl6,Ccl20,Nfkbia,Cldn4,Mmp9,Hspa8,Icam1 and Jun were expressed.3.After LPS induces inflammation in epididymal epithelial cells,the expression of IL-1beta and TNF-alpha in the cells treated with leptin was increased by ELISA,while the expression of genes NF-kappa B p65,NF-kappa B p50,IL-1?and TNF-alpha in the cells treated with leptin was increased by q PCR.After PDTC pretreatment with LPS,the expression of IL-1beta and TNF-alpha in the cells treated with leptin was significantly lower than that in the LPS+Leptin group.At low level,the expression levels of gene NF-kappa B p65,NF-kappa B p50,IL-1?and TNF-alpha also decreased significantly.ConclusionChronic epididymitis and elevated leptin can decrease sperm motility and normal morphology.The expression of NF-?B inflammatory pathway gene and protein in epididymitis model rats increased,suggesting that NF-?B pathway plays a role in epididymitis.Leptin may affect the development of epididymic epithelial cell inflammation through the NF-k B pathway.