| Assisted reproductive technology(ART)is a crucial infertility treatment in modern reproductive medicine.However,as the success rate of ART treatment is still low,obtaining high quality sperm is important for enhance the success rate of ART.In the ART process,after obtaining semen from a male infertile patient,sperm sorting is performed first.High quality sperm is obtained by removing impurities and low-quality sperm.The sorted sperm is successfully capacitated in vitro in order to penetrate and fertilize the ovum.Therefore,optimizing the sperm sorting and in vitro capacitation process,two important research areas in the field of reproductive medicine,are vital for enhance the sperm quality.Previous studies demonstrated that,to compare with traditional sperm sorting methods,the microfluidic chip was easy to operate,less damaging to sperm.For sperm in-vitro capacitation,sperm culture fluid medium has always been a key part.Previous studies have tried to add various supplements to the culture fluid medium in order to enhance the efficiency of in vitro capacitation.Most of microfluidic chips were more complicated in design and costly.There were still few sperm culture supplements in studies,and the effect of them in sperm capacitation was not stable.Cytokines secreted by cumulus-oocyte complex(COCs)during internal fertilization.There were four parts in this study.In part one and two,we used our home-made microfluidic chip to sort sperm and observe the impacts on sperm quality.In part three and four,the Human Tubal Fluid(HTF)medium was modified by adding mouse COCs,and the sperm quality after in vitro capacitation was analyzed.Part 1Methods: Using polymethyl methacrylate(PMMA)as the material,the microfluidic chips was made according to functional requirements.Semen samples were obtained from male infertile patients.Semen samples were respectively placed in the non-toxic sterilized centrifuge tube and microfluidic chip for 1h,2h,3h,4h and then the changes in sperm motility of the two groups were compared at each time point.Sperm placed in the microfluidic chip were sorted for 40 min,60min,and 80 min,respectively and the changes of their parameters were analyzed at each time point.In order to evaluate the impacts of different seletctive approaches on the quatity of sperm,the samples were sorted by the microfluidic chip,swim-up and density gradient centrifugation,respectively,and then the changes of sperm parameters were analyzed before and after sorting.Results: There was no significant difference in sperm motility and percentage of progressive sperm between the non-toxic sterilized centrifuge tube and the microfluidic chip after placing for 1h,2h,3h and 4h(P>0.05).Compared with pre-sorting,sperm motility and percentage of progressive sperm increased significantly after sorting for 40 min,60min and 80min(P<0.01),and sperm concentration decreased significantly(P<0.01).However,sperm concentrations at60 min and 80 min were notably higher than that at 40min(P<0.05).Compared with the swim-up and density gradient centrifugation,sperm motility and percentage of progressive sperm sorted by the microfluidic chip were remarkably elevated,while sperm concentration and DFI were significantly decreased(P<0.05).Conclusion: Our homemade microfluidic chips are non-toxic to sperm and can obtain high-quality sperm as evidenced by higher motility and less DNA damage,which is more suitable for application in Intracytoplasmic sperm injection(ICSI).However,the disadvantage of low sperm concentration after sorting is still to be improved.Part 2Methods: As the disadvantage of low sperm concentration after sorting,the microfluidic chip was modified.Semen samples were obtained from male infertile patients.The samples were sorted by the two microfluidic chips(pre and post modification),respectively,and then the changes of sperm parameters were analyzed before and after sorting.In order to evaluate the impacts of different seletctive approaches on the quatity of sperm,the samples were sorted by the microfluidic chip,swim-up and density gradient centrifugation,respectively,and then the changes of sperm parameters were analyzed before and after sorting.Results: Compared with the pre-modified microfluidic chip,sperm concentration sorted by the post-modified microfluidic chip were significantly increased(P<0.05),while sperm motility,percentage of progressive sperm,DFI and percentage of normal morphology sperm were no significant difference(P>0.05).Compared with the swim-up and density gradient centrifugation,sperm motility,percentage of progressive sperm,the curvilinear velocity(VCL),the straight line velocity(VSL),the average path velocity(VAP)sorted by the microfluidic chip were significantly increased,while sperm concentration and DFI were significantly decreased(P<0.05).Conclusion: Sperm concentration sorted by the modified microfluidic chip was notably higher than that before the modification.Compared with the density gradient centrifugation and the swim-up,sperm concentration sorted by the modified microfluidic was still low.However,the modified microfluidic chips provide the possibility to improve sperm quality during in-vitro fertilization(IVF)in some infertile patients.Part 3Method: Eight-week-old ICR mouse COCs were added to HTF medium and crushed to obtain the post-modified HTF medium.Twelve-week-old ICR mouse sperm was capacitated in vitro with the pre-modified and post-modified HTF medium.The changes of the sperm parameters of the two groups were analyzed;Twelve-week-old ICR mouse sperm after in-vitro capacitation was added to combine with the ova of mouse,and then the changes of in vitro fertilization rate and the number of sperm combined with the zona pellucida of oocytes of the two groups were analyzed.Results: There was no significant difference in sperm motility,percentage of progressive sperm,VCL,VSL,VAP and DFI between the two groups(P>0.05).(2)To compare with the pre-modified HTF medium,after in-vitro capacitation with the post-modified HTF medium,in vitro fertilization and the number of sperm combined with the zona pellucida of oocytes increased significantly(P<0.01).Conclusion: Compared with the pre-modified HTF medium,in-vitro capacitation with the post-modified HTF medium can improved the fertilization ability of sperm.Part 4Methods: Mouse sperm were capacitated with the pre-modified and post-modified HTF medium was collected.Isobaric tags for relative and absolute quantitation(i TRAQ)were used to detect the differentially expressed proteins in the two groups.The expression of SERPINA5 was analyzed by Western blot(WB).The post-modified HTF medium was obtained with the same approaches in part 3.Twelve-week-old ICR mouse sperm was capacitated in vitro with the post-modified HTF medium and divided into three groups.SERPINA5 antibody was added in the first group,BETA-ACTIN antibody was added in the second group,and then the changes of in vitro fertilization rate and the number of sperm combined with the zona pellucida of oocytes of the three groups were analyzed;the changes of sperm parameters of the three groups were also analyzed.The ovarian tissue of Eight-week-old ICR mouse was detected with immunohistochemical staining to observe the presence of SERPINA5 protein in COCs.Twelve-week-old ICR mouse sperm was capacitated in vitro with HTF medium with different concentrations of recombinant mouse SERPINA5 protein added.Sperm after in-vitro capacitation was added to combine with the ova of mouse,and then the changes in number of sperm bound to the zona pellucida of the oocytes in the all groups were analyzed.Results: Compared with the pre-modified HTF medium,there were 39 proteins with increased expression and 31 proteins with decreased expression in sperm samples of mouse after in-vitro capacitation with the post-modified HTF medium.The expression of SERPINA5 in sperm after in-vitro capacitation with the post-modified HTF medium was significantly increased(P<0.05).Compared with no antibody group and adding BETA-ACTIN antibody group,the in vitro fertilization and the number of sperm combined with the zona pellucida of oocytes was significantly decreased after adding SERPINA5 antibody(P<0.05).Compared with no antibody group and adding BETA-ACTIN antibody group,sperm motility,percentage of progressive sperm,VCL,VSL,VAP and DFI were no significant difference(P>0.05).SERPINA5 protein was distributed in mouse COCs.Compared with no SERPINA5 protein group,in vitro fertilization and the number of sperm combined with the zona pellucida of oocytes increased significantly after adding 1?g/ml or 2 ?g/ml recombinant mouse SERPINA5 protein to HTF medium(P<0.01).Conclusion: The expression of SERPINA5 has an important effect on the improvement of mouse sperm fertilization ability,but the molecular mechanism remains to be further studied. |
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