Clinical And Experimental Study On The Treatment Of Varicocele-related Infertility With Shengjing Huoxue Recip

Although VC is not a determinant of male infertility,it is closely related to male infertility status and is currently recognized as one of the most common causes of male infertility.The expectant treatment which lack of special effects,contain Octapenins such as Aescuven forte and Antioxidant drugs such as Vitamins C,E,Levocarnitine,Co-Q10,etc.The surgical treatment requires strict control of surgical indications,which including open surgery,laparoscopic,microsurgery,embolization,etc.There is uncertainty about the efficacy of surgery,and as an invasive treatment,which is not easy to be accepted and has a certain recurrence rate and complications.TCM has a good curative effect in the treatment of VC-associated infertility.For patients with unilateral varicocele,TCM treatment can also further improve the function of testicular and epididymis of the healthy side.On the basis of professor Li Yue qing,the academic thought of male infertile kidney deficiency and blood stasis and the "Medicalprin·Sterility"-"ZhongHeZhongZi Direction",We founded the ShengJingHuoXue Direction,Which has remarkable effective in the treatment of VC-associated infertility.However,there has no rigorous randomized controlled studies to evaluate its exact efficacy.Meanwhile,the treatment mechanisms of the ShengJingHuoXue Direction have not been deeply explored.ObjectiveWe conducted the clinical trial to objectively evaluate the clinical efficacy of ShengJingHuoXue Direction on VC-associated infertility.At the same time we designed animal experimentation to explore the effect of ShengJingHuoXue Direction on spermatogenic function of experimental left VC rats and discusse the treatment mechanisms about ShengJingHuoXue Direction deeply.MethodsClinical research:A randomized controlled trial was used,80 patients with VC-associated infertility were divided into control and treatment groups using random digital table method.The control group was given Maizhi Ling tablets+Wuzi Yanzong pill,and the treatment group was given ShengJingHuoXue Direction.The treatment was performed for 12 weeks.Before and after the treatment,the semen volume,sperm concentration,prorsad running sperm(PR),total motility(PR+NP),sperm DNA fragmentation index(DFI),TCM syndrome scores and venae spermatica diameter of the two groups were observed.Follow-up for 6 months,the spouse pregnancy was recorded.Main efficacy indicators:① spouse pregnancy.Secondary efficacy index:① TCM syndrome score;② venae spermatica diameter;③ semen volume,sperm concentration,forward movement(PR)sperm percentage,total sperm motility(PR+NP),sperm DNA fragmentation rate(DFI).The visits were conducted on 0 day,4 weeks±3 days,8 weeks ±3 days and 12 weeks ±3 days respectively to record observational indicators,at the sametime the safety and adverse events were recorded.The case report forms were filled,the shedding and removal of cases were evaluate strictly during each visit,and all the data were counted finally.Experimental research:Forty male SD rats were fed adaptively for one week before mold making.According to body weight,the rats was divided into 4 groups random numbers method,10 rats each group,namely sham group(NVC group),experimental varicocele model group(VC group),Maizhi Ling group(control group,M+VC group),and ShengJingHuoXue Direction group(treatment group,S+VC group).The VC group,M+VC group and S+VC group were prepared experimental varicocele model by partial constrictive left renal vein+collateral ligation(Improved Turner method),and the rats in NVC group were isolated the left renal vein only.Drug gavage was started 8 weeks after surgery,on the does of lml/100g/d.The treatment group was infused at 0.36g/ml with ShengJingHuoXue Direction;the control group at 6 mg/ml with Maizhi Ling.The model and sham groups were perfused with an equal volume of distilled water by the upper method。For a total of 48d.The rats were killed to observe sperm quality,serum hormones,histopathological morphology of testiculus,the oxidative stress of testiculus(SOD by WST-1,CAT by Visible photo method,GSH by Micro plate method,8-OHdG by ELISA and MDA by TBA).The protein expression of NF·E2-related factor(Nrf2)and glutathione peroxidase-4(GPx4)in rat testicular tissue were measured by Westblot.The mRNA transcribe of NF·E2-related factor(Nrf2),glutathione peroxidase 4(GPx4),and 8-hydroxyguanine DNA glycosidasel(OGG1)in rat testicular tissue were measured by Real-Time PCR.ResultsClinical research:Finally,35 cases were completed on the treatment group,total efficiency rate 88.6%,34 cases were completed on the control group,total efficiency 70.6%.Overall efficacy comparisons between the two groups showed significant differences(P<0.05).The spouse pregnancy rate in the control group was 14.7%and 25.7%in the treatment group.The spouse pregnancy rate in the treatment group was higher than that in the control group,but there was no significant difference between the two groups(P>0.05).The sperm concentration,PR sperm,(PR+NP)sperm and sperm DFI improved significantly than before treatment(All P<0.05);The treatment group improved better than the control group(All P<0.05)in sperm concentration,(PR+NP)sperm and sperm DFI;The TCM syndrome scores decreased significantly(All P<0.05),but there was no significant difference between the two groups(P>0.05).The trend of venae spermatica narrowing was observed in both groups after treatment,However,there was no significant difference(All P>0.05).Experimental research:The weights of left testis in the VC group were significantly less than the NVC group(P<0.01),while in the VC+S group were significantly higher than those in the VC group(P<0.01).Compared with NVC group,serum T in VC group decreased significantly(P<0.05),but the serum FSH and LH rised significantly(P<0.05);Compared with the VC group,the serum T in VC+S group increased significantly(P<0.05),the serum FSH and LH decreased significantly(P<0.05).Compared with the NVC group,in the VC group the sperm concentration and motility decreased significantly(P<0.01),while the sperm DFI increased significantly(P<0.01);Compared with the NCV group,in the VC+S group the sperm concentration and motility increased significantly(P<0.01),the sperm DFI decreased significantly(P<0.01).Compared with NVC group,in the testiular tissue of VC group,the SOD、CAT viability and GSH content were decreased significantly(P<0.05),MDA,8-OHdG content rised significantly(P<0.05).Compared with VC group,in the testiular tissue of VC+S group,the SOD、CAT viability and GSH content were rised significantly(P<0.05),MDA,8-OHdG decreased significantly(P<0.05).Compared with the NVC group,the seminiferous tubules in the VC group were poorly organized and disordered,with a large reduction of spermatogenic epithelial cells in all levels and compared with the VC group sperminiferous epithelium in the VC+S group was significantly mild.The testicular Nrf2 protein expression in VC group was significantly higher than the NVC group(P<0.05),while the testicular GPx4 protein expression was significantly lower than the NVC group(P<0.05).In the VC+S group,the testicular Nrf2 protein expression was significantly higher than the VC group(P<0.05),also the testicular GPx4 protein expression was higher than the VC group(P<0.01).The transcript levels of Nrf2 mRNA and GPx4 mRNA in the VC group were significantly lower than those in the NVC group(P<0.05).The transcript levels of Nrf2 mRNA and GPx4 mRNA was significantly higher in the VC+S group than in the VC group(P<0.05).The testicular OGG1 mRNA transcript levels in the VC group was significantly lower than that in the NVC group(P<0.05),Higher OGG1mRNA transcript levels was observed in the VC+S group compair with the VC group(P<0.05).Conclusions1.ShengJingHuoXue Direction can significantly improve semen volume,semen concentration and sperm vitality in male infertile patients,reduce sperm DFI,improve TCM syndrome score,and improve the pregnancy rate of spouses.2.The rat spermatogenic dysfunction model caused by experimental varicocele was successfully prepared,which can cause oxidative stress in testicular tissue,lipid peroxidation and DNA oxidative damage.3.ShengJingHuoXue Direction can improve the testicular organ index of experimental varicocele rats,improve sperm quality,reduce the oxidative stress of testicular tissue,and have a certain repair effect on the structure and function of sperminiferous epithelium,the improvement of reproductive endocrine levels..4.ShengJingHuoXue Direction can regulate the expression of Nrf2,GPx4 protein and the transcript levels of Nrf2,GPx4,OGG1 mRNA in testicular tissue,reduce the lipid peroxidation of spermatogenic cells by regulating the Nrf2-GPx4 pathway,avoid plasma membrane damage in spermatogenic cells.Meanwhile,it can regulate OGG1mRNA transcript levels in BRE and accelerate DNA repair in oxidatively damaged spermatogenic cells.In conclusion,ShengJingHuoXue Direction can significantly improve the semen quality,reduce semen DFI and improve the spouse pregnancy rate of infertile patients with varicocele.Some of the mechanisms are related to the regulation of the Nrf2/GPx4 pathway to reduce lipid peroxidation in spermatogenic cells,and to regulate the BRE to reduce DNA oxidatively damage in spermatogenic cells.