Study Of Protein Oxidative Stress In Human Follicular Fluid, Embryo Culture Media And Frozen Semen

With the establishment and development of radical theory, people have foundthat certain oxygen metabolites and their certain derivatives can damage the body.The chemical properties of these substances are more active than that of oxygen.These substances are named as reactive oxygen species (ROS). In normal condition,the production and elimination of ROS can maintain dynamical equilibrium. But incertain pathological conditions and aging, ROS increases or the cell anti- oxidationmechanism suffers injury, which can cause the accumulation of ROS and theoxidative damage of macromolecular substances such as deoxyribonucleic acid(DNA), lipid and protein. The oxidized damage of DNA can cause mutations ordeletions of mitochondrial DNA and nuclear DNA. Lipid peroxidation of cellmembrane can change membrane fluidity. But protein oxidation causes certainimportant enzyme to be inactived and protein function to suffer injury.People already discovered the existence of ROS in follicular fluid. Low level ofROS plays an important role in normal growth of oocytes. However, high level ofROS has adverse effects on oocyte maturation and the following embryonicdevelopment. The researches also discovered oxidative stress involving in theoccurrence of defects in embryonic development. High level of ROS in embryoculture medium of the first day has adverse effects on embryonic development. When sperm is frozen and in a state of heat stress, the level of ROS will increaseremarkably. These results suggest that human follicular fluid, embryo culture mediumand frozen semen exist in the phenomenon of ROS-induced oxidative stress. Previousstudies mainly pay attention to ROS-mediated lipid peroxidation damage, but recentstudies shfow that oxidative modification of protein couldn't be ignored. To date, inhuman follicular fluid, embryo culture medium and frozen semen, ROS mediatedprotein oxidative damage has not been reported. So does protein oxidative stress existin the above systems? Whether its degree can lead to an adverse impact? On the basisof some reproductive researches about ROS and human other organization studiesabout protein oxidation, we take advanced oxidation protein products (AOPP) for thetarget of protein oxidative damage, observe whether protein oxidation exists inhuman follicular fluid, embryo culture medium and frozen semen, and explore theeffects of protein oxidation on oocytes and early embryonic development.Partâ… Relationships between the Level of Protein Oxidation inHuman Follicular Fluid and Outcome Parameters of IVF-ETObjective:To explore relationships between the level of protein oxidation in humanfollicular fluid and outcome parameters of IVF-ET.Methods:1. Samples: 64 samples of follicular fluid were obtained from female infertilitypatients receiving IVF-ET treatment in reproductive center of Nanfang Hospitalfrom July 2005 to April 2006.Case selection criteria: the age from 25 to 38 yearswith a mean age of 32.41±3.17 years; simple tubal factor infertility; conventionalIVF-ET Treatment; without endometrial diseases and the thickness of endometriumabove 8.0 millimeter on HCG date. Grouping: according to the number of retrievedoocytes, samples were divided into three groups of below 8, from 8 to 15 and over 15. According to age, samples were divided into three groups of below 30 years, from 30to 35 years and over 35 years.2. Methods: AOPP, representing protein oxidative level, was determined byspectral analysis with absorbance at 340nm in acidic condition.3. Statistical methods: Carry on the analysis using spss10.0 statistics software,partial correlation analysis, one-way ANOVA analysis and independent samplest-test.Results:1. The level of AOPP correlated inversely to the proportion of mature oocytes(r=-0.401,P=0.001), fertilization rate (r=-0.257,P=0.045),cleavage rate(r=-0.290,P=0.024) and good embryo rate (r=-0.520,P=0.000) significantly.2. The difference of the level of AOPP has statistics significance (F=3.851, P=0.027)in different groups of the number of retrieved oocytes. The level of AOPP was thelowest in the group of oocytes from 8 to 15 and the highest in the group of oocytesbelow 8.3. The level of AOPP in the non-pregnancy group was significantly higher thanthat of the pregnancy group (t=3.665,P=0.001).4. The difference of the level of AOPP has statistics significance(F=15.919,P=0.000) in different age groups. The level of AOPP was the lowest in thegroup of age over 35years and the highest in the group of age below 30 years.Conclusions:1. Protein oxidative stress in follicular fluid exists in IVF-ET cycle.2. High level of AOPP may have adverse effects on oocytes and the earlyembryonic development, then affect outcome of IVF-ET.Partâ…¡Relationships between the Level of Protein Oxidation inHuman Embryo Culture Media of the First Day and the EarlyEmbryonic DevelopmentObjective: To explore relationships between the level of protein oxidation in human embryoculture media of the first day in IVF-ET circle and the early embryonic development.Methods:1. Samples: Samples of embryo culture media of the first day were obtainedfrom female infertility patients receiving treatment of routine IVF-ET andintracytoplasmic sperm injection-embryo transplantation (ICSI-ET) in reproductivecenter of Nanfang Hospital from July 2005 to November 2005. Grouping: accordingto age, samples were divided into three groups of below 30 years, from 30 to 35 yearsand over 35 years.2. Methods: AOPP, representing protein oxidative level, was determined byspectral analysis with absorbance at 340nm in acidic condition.3. Statistical methods: Carry on the analysis using spss10.0 statistics software,partial correlation analysis, one-way ANOVA analysis and independent samplest-test.Results:1. In routine IVF circle, when the density of AOPP in embryo culture media ofthe first day is below 3.2μmol/l,it did not correlate to fertilization rate(r=0.137,P=0.863), cleavage rate (r=0.623,P=-0.377), fragmentation rate(r=-0.156,P=0.844) or the embryo formation rate more than six cells(r=-0.444,P=0.556);when the density is over 3.2μmol/l, it also did not correlate tofertilization rate(r=-0.226,P=0.338), cleavage rate(r=0.202,P=0.392), fragmentationrate (r=-0.232,P=-0.325) or the embryo formation rate more than six cells(r=0.008,P=0.975).2. In ICSI circle, when the density of AOPP in embryo culture media of the firstday is below 3.2μmol/l,it did not correlate to fertilization rate (r=-0.323,P=-0.791),cleavage rate (r=0.014,P=0.991), fragmentation rate (r=0.238,P=0.847) or theembryo formation rate more than six cells (r=-0.029,P=0.982); when the density isover 3.2μmol/l, it correlated negatively to fertilization rate (r=-0.472,P=0.031),fragmentation rate (r=-0.482,P=0.027) and the embryo formation rate more than six cells (r=-0.548,P=0.010),but it did not correlate to cleavage rate(r=-0.096,P=-0.679).3. In routine IVF circle, the level of AOPP in the non-pregnancy group wassignificantly higher than that of the pregnancy group (t=-3.160,P=0.004); thedifference of the level of AOPP has statistics significance (F=19.395,P=-0.000) indifferent age groups. The level of AOPP was the lowest in the group of age over 35years and the highest in the group of age below 30 years.4. In ICSI circle, the level of AOPP in the non-pregnancy group wassignificantly higher than that of the pregnancy group (t=-2.421,P=0.022); thedifference of the level of AOPP has statistics significance (F=3.975,P=0.031) indifferent age groups. The level of AOPP was the lowest in the group of age over 35years and the highest in the group of age below 30 years.Conclusions:1. In IVF-ET circle, phenomenon of oxidative stress exists in embryo culturemedia of the first day.2. In ICSI circle, high level of AOPP may have adverse effects on the quality ofembryo and outcome of IVF.3. In routine IVF circle, high level of AOPP may have not effect on the qualityof embryo, but may adverse effects on outcome of IVF.Partâ…¢Effects of Cryopreservation and Incubation onthe Level of Oxidative Stress of Human SpermObjective:To explore effects of cryopreservation and incubation on the level of oxidativestress of human sperm.Methods:1. Samples: 60 normal semen samples were obtained from male patientsreceiving treatment of IVF - ET because of female factors in reproductive center ofNanfang Hospital. For each sample, after gradient centrifugation, we obtainspermatozoon, add human tubal fluid (HTF) to maintain the density of sperm in (20～30)×10⁶/ml, take 2.5 ml to divide into five groups equally including untreatedcontrol group, cryopreservation blank assay group, cryopreservation sample group,incubation blank assay group and incubation sample group.2. Methods: AOPP, representing protein oxidative level, was determined byspectral analysis with absorbance at 340nm in acidic condition. Malondialdehyde(MDA), representing lipid peroxidation, was analyzed by thiobarbituri (TBA) acidreaction.3. Statistical methods: Carry on the analysis using spss10.0 statistics softwareand one-way ANOVA analysis.Results1. The difference of the level of AOPP(F=19.994,P=0.000) and MDA(F=31.837,P=0.000) had statistics significance in different groups.2. Compared with untreated control group and cryopreservation blank assaygroup, cryopreservation sample group had significantly higher level ofMDA(P=0.000), while the level of AOPP had no difference significantly.3. Compared with untreated control group and incubation blank assay group,incubation sample group had significantly higher levels of MDA(P₁=0.000;P₂=0.000) and AOPP (P₁=0.000; P₂=0.000).4. The AOPP and MDA's levels of incubation sample group were higher than thatof cryopreservation sample group (AOPP: P=0.000; MDA: P=0.046).Conclusions:1. Oxidative damage of human sperm exists in cryopreservation and incubation.2. The main oxidative damage of cryopreservation is lipid peroxidation, andincubation can not only induce lipid peroxidation, but also protein oxidation.