Utility Of Oxidative Stress Test In Male Infertility Clinic And Assisted Reproductive Technology

PART ONECharacterization of oxidative stress status evaluated by ROS detection in whole semen and human sperm motility assay with seminal plasmaObjective:To investigate the distribution of reactive oxygen species(ROS) levels in whole semen collected from infertile males in China and the toxicity of seminal plasma with oxidative stress to sperm motility.Methods:ROS levels of semen samples collected from 451 Chinese male factor infertility (MFI) patients and 50 fertile donors were detected by chemiluminescence assay. Human motility assay was performed to observe the toxicity of 3 groups of seminal plasma with low,median and high ROS levels.Results:ROS levels of whole ejaculates from Chinese male factor infertility patients fit the skewness distribution.ROS-positive rate is 46.6%,and 24.3%of the ROS-positive samples may mainly result from leukocyte-contaminating.ROS level was related to sperm motility and morphology.The toxicity of seminal plasma was related to the extent of oxidative stress of primitive semen.Conclusion:The detection of ROS level by measuring the whole ejaculates can really reflect the function of sperm.Kinetic parameters,and morphology were related to ROS level of whole ejaculates.Besides ROS generation,oxidative stress may affect the components of seminal plasma which have eytotoxicity to sperm motility. PART TWOA novel technology to evaluate the integrity of spermatozoa membraneObjective:To develop a novel technology using membrane-permeant DNA stain——Transgreen and propidium iodide(PI)——to evaluate the integrity of sperm membrane.To develop a novel technology to evaluate the integrity of sperm membrane using fluorescent enzyme-labeling instrument.Methods:The technology to evaluate the integrity of sperm membrane using Transgreen/PI was set up.The results of detection with Transgreen/PI and LIVE/DEAD Sperm Viability Kit were analyzed.The toxic action of Transgreen and PI on sperm motility was observed.Fluorescent enzyme-labeling instrument was used to detect sperm viability.A new index "Viable coefficient(VC)" was used to reflect sperm viability.The reliability and validity of this technology were analyzed.Results:The correlation coefficient between the results of Transgreen/PI and LIVE/DEAD Sperm Viability Kit is 0.927,P＜0.0001.There is no significant difference(P＞0.05) between the results of assessment of sperm viability by different sequence of application of Transgreen and PI.2 h after Transgreen and PI administered,there was no prominent impact on motion parameters of sperm.The reliability of the technology using fluorescent enzyme-labeling instrument was greater than hand-counting.Sperm concentration did not affect the value of VC.There was strong correlation between VC and viability.Conclusion:The dual association Transgreen/PI procedure possesses good repeatability,high sensitivity,and real-time detection same as SYBR-14/PI.Besides,it is easily handled and much cheaper,1%cost compared to SYBR-14/PI.The combination of Transgreen and PI was determined to be an effective tool to evaluate the integrity of sperm membrane.A novel technology using fluorescent enzyme-labeling instrument and a new index VC can use for sperm viability assessment.It is more reliable than hand-counting and much cheaper than flow cytometry. PART THREEChinese Traditional Medicine Combined with Intracytoplasmic Sperm Injection to Treat Severe Oligospermatism and AzoospermiaObjective:To observe the advantage of Bushen Shengjing Decoction(BSSJD),a Chinese herbal formula,which can reduce semen reactive oxygen species(ROS) level and improve the quality of sperm and the outcome of intracytoplasmic sperm injection (ICSI) cycles.Methods:A total of 100 subjects with severe oligospermatism and azoospermia were enrolled.50 subjects were assigned to Group A,received BSSJD.The other 50 subjects were assigned to Group B as control.The density,motility and semen ROS levels were compared before and after BSSJD.After 3 months therapy,83 patients of the two groups received ICSI.The number MII eggs,the rates of fertilization, cleavage,available embryo and clinical pregnancy of two groups were compared.Results:After BSSJD therapy,the semen ROS levels were decreased and the density, motility of sperm increased(P＜0.05).The rates of fertilization and clinical pregnancy in Group A are higher than those in Group B.Conclusion:BSSJD has the effect of decreasing ROS level in semen and improving the quality of sperm.It can also increase fertilization rate in ICSI cycles.