Effect Of Varicocele On Reproductive Hormone,Seminal Plasma Biochemistry And Semen Quality In Infertile Patients And The Experimental Study On The Effects Of HIF-1? On Autophagy,apoptosis And Testerone Metabolism In TM3 Cells Under Hypoxia

Objective We retrospectively analyzed the effect of VC on reproductive hormone,seminal plasma biochemistry,semen quality,and combined with the experiment exploring the effects of HIF-1?on autophagy,apoptosis and testosterone metabolism of Leydig cells under hypoxic conditions to provide the basis for the diagnosis and treatment of VC and for studying possible mechanism of VC damaging male fertility.Methods Clinical study:Data were collected from out-patients at Renji hospital of Shanghai Jiaotong university from October 2017 to November2019,including 58 Varicocele patients and 67 infertile men without Varicocele.We compared the general information,endocrine level,seminal plasma biochemistry and semen quality among them.Experiments:TM-3 cells were cultured in oxygen at the concentration of1%for different hours.We detected the viability of the TM-3 cells by CCK-8 assay,determined the levels of protein and m RNA of HIF-1?by Western Blot and q PCR.TM-3 cells were cultured in oxygen at the concentration of 1%or 20%for 16 hours after transfected with HIF-1?si RNA or Control si RNA.The expression of LC3B I,LC3B II,p62,Bax,Bcl-2,PARP,cleaved-PARP were detected by WB.The levels of m RNA of St AR,CYP11A1,HSD3B2 were deceted by q PCR.The pretreatment groups were treated with Rapamycin or 3-Methyladenine dilution for 1 hour,and then placed in 1%O₂for 16 hours.Results Clinical study:1.There was no significant difference in age,height,BMI,testicular volume and testicular atrophy index between VC patients and non-VC patients(P>0.05).2.PRL level in peripheral blood of VC patients was significantly lower than that of patients without VC(P<0.05),and there was no significant difference in other reproductive hormones,seminal plasma biochemistry and semen quality between two groups.3.PRL level in peripheral blood of Clinical VC patients was significantly lower than that of patients without VC(P<0.05),and there was no significant difference in other reproductive hormones,seminal plasma biochemistry and semen quality between two groups.4.There was no significant difference in reproductive hormones,seminal plasma biochemistry and semen quality between Subclinical VC patients and non-VC patients(P>0.05).Experiments:1.At 16 hours,the viability of the TM-3 cells began to decrease significantly and the expression of HIF-1?began to increase with the prolonged time in oxygen at a concentration of 1%(P<0.05).2.Compared with the 20%normoxic group,the expression of LC3B II,Bax and cleaved-PARP increased and the expression of p62 and Bcl-2decreased in the 1%hypoxia group.The St AR and CYP11A1 m RNA level of TM3 cells significantly decreased after 1%hypoxia treatment compared with normoxia(P<0.05),but the HSD3B2 m RNA was slightly decreased.3.The expression of HIF-1?was decreased after being transfected by HIF-1?si RNA in the 1%hypoxia group,confirming the successful transfection.4.The level of LC3B II/LC3B I,Bax/Bcl-2 decreased and the level of p62and Bcl-2 increased in hypoxia+si-HIF-1?group than that in hypoxia+si-NC group,and the level of St AR m RNA and CYP11A1 m RNA significantly increased(P<0.05).5.The level of St AR m RNA and CYP11A1 m RNA in 3-MA+hypoxia group significantly decreased,while the expression of St AR m RNA significantly increased in Rapa group(P<0.05).Conclusion Clinical study:In male infertility,the effect of Subclinical Varicocele on fertility is not significant and the level of PRL in patients with Clinical Varicocele was relatively low.Clinical Varicocele has no significant effect on other reproductive hormones,seminal plasma biochemistry and semen quality,which may be related to the distribution of VC degree.Experiments:Hypoxia suppresses the viability of TM-3 cells and increases the expression of HIF-1?.The hypoxia can induce the autophagy,apoptosis and inhibit the expression of testosterone metabolism-related genes of TM-3 cells by HIF-1?.The activation of autophagy could alleviate the negative effect of hypoxia on St AR expression and promote the synthesis of testosterone.The effect of Varicocele on serum testosterone level was not significant,possibly because cell alleviates the negative effect of Varicocele on synthesis of testosterone through autophagy.