| Objective About 8% to 12% of couples in the world suffer from infertility.Among them,40% to 50% are male infertility due to the “male factor”.Semen abnormalities are the most common,and teratozoospermia accounts for at least1/3,the normal morphology of sperm is the key to the determination of sperm and egg.The normal fertilization process in sperm depends on the normal sperm morphology.The pathogenesis of teratozoospermia is complicated and may be associated with chromosomal abnormalities,gene mutations or deletions,hormonal disorders,immune abnormalities,infections,environmental deterioration,exposure to toxic radiation,varicocele,excessive reactive oxygen species(ROS),oxidative stress,and sperm DNA damage and other factors,mostly combined with sperm motility,suggest that harmful factors activate the signal channel receptor on the sperm surface,alter the sperm structure through signal transduction pathways,and cause abnormal sperm motility and morphology,including excessive ROS,Oxidative stress and sperm DNA damage are currently hot topics.However,at the moment,there is no unified understanding of what kind of signal pathways the process information will pass through.Finding the mechanism that leads to the abnormal morphology of spermatozoa will help to find a therapeutic treatment and make the infertile patients with teratozoospermia acquire the ability to produce offspring.The history of treatment of infertility by Chinese medicine is more than2,000 years old.Male infertility spermatozoa is an ancient "no child" category.The tutor thinks the disease is a disease with complex pathological mechanism.Kidney deficiency and blood stasis is one of its important pathological foundations.It is identified as the syndrome of kidney deficiency and blood stasis.Various factors can lead to deficiency of kidney yang,lower coke deficiency,lack of warm phlegm,lack of genital or kidney yin deficiency,yin deficiency,seizure of semen,burning of sperm or "qi deficiency" to promote blood flow,yang loss,no temperature Blood,blood flow is slow and stagnant,blood flow is easy to make the accumulation of pathological products damage the micro-environment of sperm survival,resulting in increased deformity rate,affecting sperm function,resulting in infertility.Therapeutic use of Bushenhuoxue method is to treat with " Bushen Huoxue Fang",in order to improve the micro-environment of sperm survival,increase blood flow,fight against ischemia and anoxia,oxidative stress,to protect the sperm from damage and treat the teratozoospermia.This study will use Bushen Huoxue Prescription for the treatment of patients with male infertility due to kidney deficiency and blood stasis.The parameters of sperm and sperm DNA integrity(DNA Fragmentation Index,DFI)before and after treatment are measured by Computer semen parameters analysis(CASA)and Flow Cytometry(FCM).understanding 0f the effect of Bushen Huoxue Prescription on Sperm of Male Infertility Patients with Deficiency of Kidney Deficiency and Blood Stasis;Western blotting(WB)was used to detect the expression of JAK2/STAT3 protein in normal and abnormal spermatozoa and the relationship between JAK2/STAT3 signal transduction pathway and sperm morphology;The Bushen Huoxue Decoction was used to treat patients with kidney insufficiency and blood stasis type male infertility sperm disease.Semen parameters and the expression of JAK2/STAT3 protein in sperms before and after treatment were detected by CASA and WB,Understanding the Effect of Bushen Huoxue Prescription on Expression of JAK2/STAT3 Protein in Sperm of of Male Infertility Patients with Deficiency of Kidney Deficiency and Blood Stasis;finally,use different concentrations of JAK2/STAT3 inhibitor(AG490)to act on the normal morphology of sperm,Detection of Inhibition of Spermatozoa After Intervention with FCM Changes in the DFI to understand the effect of inhibiting JAK2/STAT3 signaling pathway on sperm DNA integrity,and to provide a scientific basis for exploring the pathogenesis of teratozoospermia and exploring the treatment of teratozoospermia.MethodsThis topic is divided into two aspects of clinical research and experimental re1.一、Clinical research:A total of 60 patients aged from 23 to 45 years old who were diagnosed of male infertility teratozoospermic patients were collected from March 30,2016 to September 30,2017 in the Department of Reproductive Medicine of the First Affiliated Hospital of Guangzhou University of Chinese Medicine.Symptoms,given TCM syndrome differentiation "kidney deficiency and blood stasis syndrome" were included in the study.Using the randomized principle,the subjects were divided into three groups: Bushen Huoxue Fang group(A group),antioxidation therapy group(B group)and non-treatment group(C group).Group A(20 cases): Tonic of Bushen Huoxue Fang Decoction was given after dialectical addition and subtraction for a period of 12 weeks;Group B(20 cases): Daily oral administration of natural vitamin E 100 mg twice a day for 12 weeks;C Group(20 cases): blank group,no drug treatment.In the three groups of subjects,CASA was used to analyze semen routine parameters before and after12 weeks(treatment).Total sperm count,sperm concentration,total sperm motility,and sperm motility parameters were measured.Sperm morphology was calculated using the Diff-Quik method.Percentage of normal morphological spermatozoa,percentage of abnormal spermatozoa,abnormal sperm index(TZI),sperm deformity index(SDI),percentage of head defects,percentage of neck and mid-segment defects,percentage of tail main segment defects,and percentage of excess residual cytoplasm;using sperm density Gradient centrifugation was used to extract sperm,and sperm DFI was detected by FCM.After 12 weeks,follow-up and recording of the pregnancy outcomes of the three partners were included: clinical pregnancy rate,miscarriage rate,and live birth rate.二、 Experimental research(一)Randomly collected 40 patients aged from 23 to 45 years old who were diagnosed as male infertility and abnormal spermatozoa in the Department of Reproductive Medicine of the First Affiliated Hospital of Guangzhou University of Chinese Medicine from March 30,2016 to September 30,2017.Symptoms:Patients with syndromes of kidney deficiency and blood stasis were included in the study as subjects(abnormal spermatozoa group);Men with the same age at the same age as male specialists in the Department of Reproductive Medicine were selected.(Normal spouses have a history of pregnancy within the past3 months.),normal semen detection in 20 cases(normal sperm group).The two groups of subjects used CASA to analyze semen routine parameters of semen and obtain total sperm count,sperm concentration,total sperm motility,and sperm motility parameters.Diff-Quik method was used to detect sperm morphology,and the percentage of normal morphological spermatozoa and percentage of abnormal spermatozoa were calculated.Sperm abnormality index(TZI),Sperm deformity index(SDI),Percentage of head defects,Percentage of neck and mid-segment defects,Percentage of defect at the main part of the tail,and Percentage of residual residual cytoplasm;Sperm density gradient centrifugation was used to extract sperms and extract them.Total sperm protein was used to detect the expression of JAK2 protein and STAT3 protein by WB method.(二)Randomize the above-mentioned abnormal spermatozoa group of 40 patients;into the Bushen Huoxue Fang group(A group)and no treatment group(B group);A group(20 cases): To Bushen Huoxue Fang Decoction after dialectical addition and subtraction oral,the course of treatment is 12 weeks;group B(20 cases):blank group,no drug treatment;subjects of both groups were subjected to CAS analysis of semen routine parameters before and after 12 weeks(treatment)to obtain total sperm count,sperm concentration,spermatozoa Total vigor and sperm movement parameters;Diff-Quik method was used to detect sperm morphology,and the percentage of normal spermatozoa,percentage of abnormal spermatozoa,abnormal sperm index(TZI),sperm deformity index(SDI),percentage of head defects,neck and middle segments were calculated.Percentage of defects,percentage of main tail defects,and percentage of residual residual cytoplasm.Sperm density gradient centrifugation was used to extract spermatozoa and extract total sperm proteins.WB method was used to detect the expression of JAK2 protein and STAT3 protein,respectively.(三)Randomly collected 8 males(with normal pregnancy history in the past3 months)from the March 30,2016 to September 30,2017 in the male reproductive health department of the First Affiliated Hospital of Guangzhou University of Chinese Medicine.From 23 to 45 years old,normal spermatozoa were included in the study;routine collection of sperm,after treatment by density gradient centrifugation,different concentrations(0,25,50,100 μmol / L)JAK2/STAT3 inhibitor AG490 and sperm in vitro After incubation for 3 hours,sperm morphology was measured by Diff-Quik method,and the percentage of normal spermatozoa was calculated.The sperm DFI was detected by FCM.Result一、Clinical research:(一)The percentage of normal sperm morphology,total sperm motility,and sperm forward movement(PR)after treatment in the Bushen Huoxue Decoction group were higher than before treatment.The sperm DFI,percentage of head defects,percentage of tail defects,and excess cytoplasmic residual rate were higher.Before treatment,the difference was statistically significant(P<0.05).After treatment of Bushen Huoxue Decoction group,the TZI,SDI,and neck defect rate were lower than before treatment.Semen volume and sperm The concentration and sperm count were higher than those before treatment,but the difference was not statistically significant(P>0.05).The normal sperm morphology after treatment in the vitamin E group was higher than before treatment,and the tail defect rate was lower than before treatment.The differences were statistically significant.Significance(P<0.05).Patients treated with TZI,SDI,sperm DFI,semen volume,total sperm count,head defect rate,neck defect rate,and excess cytoplasmic residual rate after treatment Before treatment,sperm concentration,total sperm motility,and sperm forward movement(PR)were higher than before treatment,with no statistically significant difference(P>0.05);patients without treatment did not receive treatment.After 12 weeks,the excess cytoplasmic residual rate of the tail was lower than that of the 12 th week.The difference was statistically significant(P<0.05).The normal sperm morphology,semen volume,total sperm motility,head defect rate,and neck defect rate Increased,sperm abnormality index(TZI)and abnormal sperm index(SDI)did not change,sperm DFI,sperm concentration,total sperm count and sperm forward movement(PR)decreased compared with before treatment,the difference was not statistically significant(P> 0.05).(二)Comparison between the Bushen Huoxue Decoction group and Vitamin E group and untreated group respectively,the pregnancy rate and live birth rate of Bushen Huoxue Decoction group were higher than that of Vitamin E group and untreated group,but the difference was not statistically significant(p>0.05).二、Experimental results:(一)JAK2 and STAT3 were expressed in normal spermatozoa and teratozoospermia.The expression of JAK2 and STAT3 in normal spermatozoa was higher than that of JAK2 and STAT3 in the spermatozoa group;JAK2 protein and STAT3 protein were expressed in teratozoospermia.The teratozoospermia group was significantly weakened;the difference in protein expression between the two groups was statistically significant(p<0.05).The percentage of normal sperm morphology was positively correlated with the expression of JAK2 and STAT3,the percentage of abnormal sperm,abnormal sperm index and sperm abnormality index were negatively correlated with the expression of JAK2 and STAT3,the difference was statistically significant(p<0.05);excess residual cytoplasm and The expression of JAK2 and STAT3 were not related and the difference was not statistically significant(p>0.05)(二)JAK2 protein and STAT3 protein were expressed in the Bushen Huoxue Fang group and the untreated group;the expression of JAK2 protein and STAT3 protein in the Bushen Huoxue Decoction group was higher than that in the untreated group,and the expression level of the protein between the two groups was statistically significant(P<0.05);Two groups of patients in the Bushen Huoxue Decoction group and the non-treatment group were compared by using self-contrast method.The percentage of normal sperm morphology,sperm motility,PR,JAK2 protein and STAT3 protein expression after treatment in the Bushen Huoxue Fang group were higher than those before treatment.Statistically significant(P<0.05).Semen volume and sperm concentration were higher than before treatment,TZI,SDI were lower than before treatment,and there was no significant difference(p ≥ 0.05);The percentage of normal sperm morphology,semen volume,sperm concentration,sperm motility,PR,TZI,and JAK2 protein expression increased after 12 weeks in the untreated group.The SDI and STAT3 protein expression was lower than before treatment,with no statistically significant difference.(p ≥ 0.05).(三)The percentage of normal sperm morphology and sperm DFI after incubating with AG490 and using AG490 for 3 hours showed that the normal morphology percentage of sperm decreased with the increase of AG490 concentration,and the DFI of sperm increased with the increase of AG490,Both were statistically significant(P<0.05);however,there was no correlation between normal sperm morphology and sperm DFI by mean correlation analysis(R=0.24,P=0.14).Conclusion一、Bushen Huoxue can improve the normal sperm morphology of sperm in patients with male infertility deformity sperm,reduce sperm DFI,reduce the percentage of defects in the sperm head and the percentage of tail defects,and increase the spouse’s pregnancy rate.二、The expression of JAK2 protein and STAT3 protein in normal spermatozoa and teratozoospermia spermatozoa were higher than normal spermatozoa.Sperm abnormalities may be related to decreased expression of JAK2 protein and STAT3 protein in spermatozoa.Bushenhuoxue prescription can increase the percentage of normal sperm morphology in patients with teratozoospermia,and this process may be related to the enhanced expression of sperm JAK2/STAT3.三、The JAK2/STAT3 inhibitor AG490 can reduce the normal sperm morphology rate and the sperm DFI in vitro.The decrease of normal sperm morphology and the sperm DFI may be related to the inhibition of sperm JAK2/STAT3 signal transduction pathway system. |
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