The Study Of Hypoxia In Varicocele Induced Infertility

BackgroundInfertility, a worldwide problem, is one of the three major diseases that deeply impact human life and health now. With the development of society and the worsening of environmental pollution, infertility will become a more severe problem. As an important cause of male infertility, increasingly, varicocele (VC) attracts people’s more attention, and the related research becomes more and more in-depth. VC accounted for19to41percent of all male infertility causes, and could be corrected mostly through treatment. VC’s impact is various in different people, and it could conduct to azoospermia, oligospermia, weak sperm or abnormal sperm, and it could get fertility normally as reported. Therefor, it is special significance to investigate the mechanism of varicocele induced infertility.The mechanism of varicocele induced infertility is not clarified so far, but related research has been focused on the aspects as follows:1.Hypoxia;2. Oxidative stress;3. Obstruction of testicular microcirculation;4. Toxic effects of vasoactive substances;5. mechanism of Nitric oxide;6. Immunological factors;7. Apoptosis of spermatogenic cells;8. Changes of molecular genetics;9. Disorder of hormone secretion, and so on. In which, the effect of hypoxia has caused more and more attention of researchers, and it has become the hot spot of research in varicocele induced infertility.Hypoxia-inducible factor (HIF) is a specific factor produced by tissues when oxygen deficit and its content are proportional to the degree of hypoxia. Being a functional subunit, HIF-la was studied widely and deeply. HIF-la could enter to cell nucleus and start the transcription of target genes as EPO and VEGF when hypoxia occurs, so as to make the tissues and cells adapt to the hypoxic environment. It has been proved that varicocele in left side will conduct HIF-la overexpression in both side of the rat testis, hypoxia was existed in both testises. When hypoxia occurred, DNA of spermatogenic cells will be injured, and it will increase the number of malformed sperms. This injured spermatogenic cells should be cleared by apoptosis, but the opposite phenomenon was detected in clinic. So, the machnism of this should be deeply researched.Animal model plays an important role in study, and it is the foundation and premise for research. Excellent method of modeling will improve the rate of success and provide us with good result. Rat is our first choice for varicocele research because of the mature method of modeling and was widely used in past studies. The rat’s small size and tiny vessels enhanced level of difficulty in modeling, major vascular injury and ureter mis-ligation often appeard. Therefore, the method of modeling should be improved so as to reach higher success rate and lower complication rate.Objective1. To explore the new method to establish varicocele rat model through using microscope and discuss the advantage of the microscope in the modeling surgery with our initial experience;2. To explore the hypoxic mechanism of varicocele induced infertility by detecting the proteins which are related to hypoxia expressed in varicocele rat testises and to discuss the role played by hypoxia in varicocele induced infertility.Methods1. Modeling(1). AnesthesiaAnesthetized the rats by pentobarbital sodium with the dose of50mg/kg through intraperitoneal injection(2). Modeling by traditional operationFix the anesthetized rat in supine position; enter to the abdominal cavity through abdominal incision which is about5cm long. Find the left renal vein and ligate it together with a smooth metal probe rod which diameter is0.7mm by using4-0silk suture, the left renal vein will be partially ligated by the probe nod withdrawal; then ligate the brances of left spermatic vein to guarantee intravenous pressure, by which we establish the rat model of varicocele.(3). Modeling by microscopeAnesthesia method, rat position and principles of operation are similar to traditional operation; but partially ligate the left renal vein and ligate the branches of left spermatic vein with the using of microscope.(4). Standard of success of modelingLaparotomy was done4weeks after modeling operation, observe the left spermatic vein and the left renal; the rat will be the model of varicocele if the diameter of left spermatic vein is≥0.5mm long and no changes of left kidney in both volume and colour.(5). Data and Statistical analysisCompare the incidence of complications such as injury of renal vein or inferior vena cava and compare the success rate and mortality rate between the two groups, whether significant differences are existed.2. ImmunohistochemistryGet the testises out from the30successfully modeled rats in both sides for experiment and30normal rats of left side for control; put each testis in4%paraformaldehyde for fixation after divided into two part; embedd by paraffin in department of pathology in our hospital for preparation of immunohistochemistry; detect the expression of HIF-la, EPO and EPOR in each testis and evaluate the strength by semi-quantitative analysis; Compare difference between the two groups and analyze correlation in each twe factors through statistical analysis.3. Statistical analysisUsing Chi-square test or Fisher probabilities to compare two sample rates; Using Kruskal-Wallis H test to compare random designed multiple samples and Nemenyi test to compare each two samples; Using Wilcoxon rank sum test to compare paired samples; Using Spearman rank correlation test to test the correlation between twe samples. It is significant if P<0.05.Results1. In traditional operation group, there are totally60rats,1rats died because of injury of inferior vena cava and4died by left renal vein injury;7rats died after operation in a few days,3by intestinal obstruction,2by accident and no exact reason for other2. Four weeks after operation, left hydronephrosis was detected in4rats and left kidney necrosis in1rat;6rats’left internal spermatic vein was not obviously varicose,37rats’were varicose obviously, their diameters were more than1.0mm. Excepting2rats died by accident, the rate of successful model was63.79%, mortality rate was17.24%and complication rate was25.86%.In microscope group,30rats were undergoing operation, no inferior vena cava or left renal vein injury happened during the operation, and no hydronephrosis or kidney necrosis were appeared after four weeks.2rats’ left internal spermatic vein was not obviously varicose, but other28rats’diameters of left internal spermatic vein were≥1.0mm. The rate of successful model was93.33%, mortality rate was0and complication rate was0, too.The rate of successful model by microscope is higher than traditional group, it has significant difference (P<0.05); the mortality and complication rate was lower than traditional group, it also has significant difference (P<0.05).2. Immunohistochemical resultsHIF-1α was overexpressed in varicocele rats’ both left (experimental group1.) and right (experimental group2.) testis, not expressed in control group, it has significant difference (P<0.05), the strength in experimental group1was higher than experimental group2and higher than control group; they all have significant difference in each comparition (P<0.05).EPO was overexpressed in varicocele rats’ both left (experimental group1.) and right (experimental group2.) testis, not expressed in control group, it has significant difference (P<0.05), the strength in experimental group1was higher than experimental group2and higher than control group; they all have significant difference in each comparition (P<0.05).EPOR was overexpressed in varicocele rats’ both left (experimental group1.) and right (experimental group2.) testis, not expressed in control group, it has significant difference (P<0.05), the strength in experimental group1was higher than experimental group2and higher than control group; they all have significant difference in each comparition (P<0.05).In each experimental group, a positive correlation was between any two factors of HIF-la, EPO and EPOR (P<0.05).Conclusion1. It has great superiority to establish varicocele rat model by using microscope because it could increase success rate and reduce mortality and complication rate compared to traditional operation method.2. Hypoxia regulates the expression of EPO and EPOR through HIF-1α, suppress the apoptosis of abnormal sperm, increase the number of abnormal sperm, and affect the sperm quality.3. One side varicocele could cause hypoxia in both side of testis, impact the production of sperm, so varicocele of one side could induce infertility.