人卵泡液、胚胎培养液和冷冻精液中蛋白氧化应激的研究
发布时间：2018-09-05 16:43
【摘要】： 随着自由基理论的建立和发展，人们发现氧的某些代谢产物及其衍生的活性物质可以损伤机体，这些物质具有比氧更为活泼的化学性质，统称为活性氧(reactive oxygen species，ROS)。正常情况下，ROS的产生和清除保持动态平衡，但是在某些病理情况下和衰老时，ROS产生增加或细胞抗氧化机制受损，可造成ROS的累积，主要导致大分子物质如脱氧核糖核酸(deoxyribonucleic acid，DNA)，脂质和蛋白质的氧化损伤。DNA的氧化损伤可以导致核DNA和线粒体DNA突变或缺失，细胞膜脂质过氧化导致膜流动性改变，而蛋白的氧化损伤导致重要酶失活，蛋白功能受损。 研究发现卵泡液中存在ROS，一定水平的ROS对维持卵母细胞的正常生长发挥重要作用，ROS的过度升高对卵母细胞成熟和后续胚胎发育造成不良影响。研究还发现氧化应激参与胚胎发育缺陷的发生，第一天胚胎培养液中高水平的ROS对胚胎发育产生不良影响。精子冷冻和热应激状态会导致ROS水平显著升高。这些结果提示了人卵泡液、胚胎培养液和低温冷冻精液中存在ROS所致的氧化应激现象。以往的研究，主要关注ROS介导的脂质过氧化损伤，但近年的研究说明，蛋白质的氧化修饰不容忽视。但是目前为止，关于在人卵泡液、胚胎培养液和低温冷冻精液中ROS介导的蛋白氧化损伤的研究还未见报道，那么上述系统是否存在蛋白氧化应激现象?其程度是否会造成不良影响?本研究拟在ROS在生殖方面的研究和蛋白氧化在人类其它组织研究的基础上，以晚期氧化蛋白产物(advanced oxidation protein products，AOPP)为蛋白氧化损伤的指标，观察人卵泡液、胚胎培养液和低温冷冻精液中是否存在ROS介导的蛋白氧化损伤，并初步探讨其与卵母细胞和早期胚胎发育的关系。 第一章人卵泡液中蛋白氧化水平与IVF-ET结局参数的关系 【目的】 探讨体外受精-胚胎移植(in vitro fertilization-embryo transplantation，IVF-ET)周期卵泡液中蛋白氧化水平与IVF-ET结局参数的关系。 【方法】 1．标本来源和分组：卵泡液标本来自南方医院生殖中心2005年7月至2006年4月之间接受IVF-ET治疗的64例不孕症女性患者。病例选择标准：年龄在25～38岁，平均年龄32.41±3.17岁；单纯输卵管性因素不孕；行常规IVF-ET治疗；HCG日子宫内膜在8.0 mm以上，并且无内膜病变。根据获卵数分为三组，获卵数＜8个组16例，获卵数8～15个组32例，获卵数＞15个组16例。根据患者年龄分为三组，年龄＜30岁组11例，30岁～35岁组41例，＞35岁组12例。 2．实验方法：以AOPP作为蛋白氧化指标，采用Vitko-Sarsat等介绍的方法进行测定。 3．统计学方法：所有数据应用spss10.0统计软件进行分析，P＜0.05认为差异有统计学意义。结果以均数±标准差((?)±s)或百分率(％)表示，采用偏相关分析、One-way ANOVA方差分析和独立样本t检验。 【结果】 1．卵泡液中AOPP水平与成熟卵母细胞比例(r=-0.401，P=0.001)、受精率(r=-0.257，P=0.045)、卵裂率(r=-0.290，P=0.024)、良好胚胎形成率(r=-0.520，P=0.000)均呈显著负相关。 2．不同获卵数组之间AOPP水平有显著性差异(F=3.851，P=0.027)，其中以获卵数8～15个组AOPP水平最低，获卵数＜8个组最高。 3．非妊娠组AOPP水平高于妊娠组，有显著性差异(t=3.665，P=-0.001)。 4．不同年龄组AOPP水平差异有显著性意义(F=15.919，P=0.000)，其中年龄＞35岁组AOPP水平最高，＜30岁组最低。 【结论】 1．IVF-ET周期中，卵泡液中存在蛋白氧化应激现象。 2．卵泡液中高水平的AOPP可能对卵母细胞和早期胚胎发育造成不良影响，继而影响IVF结局。 第二章第一天胚胎培养液中蛋白氧化水平与早期胚胎发育的关系 【目的】 探讨IVF-ET周期第一天胚胎培养液中蛋白氧化水平与早期胚胎发育的关系。 【方法】 1．标本来源和分组：第一天胚胎培养液标本来自南方医院生殖中心2005年7月至11月之间进行常规IVF治疗的30例患者和单精子卵细胞内注射(intracytoplasmic sperm injection-embryo transplantation，ICSI-ET)治疗的30例患者。分组：根据受精类型分为两组，常规IVF组30例和ICSI组30例。根据不孕妇女年龄分组，常规IVF组30例中年龄＜30岁组4例，30岁～35岁组17例，＞35岁组9例；ICSI组30例中年龄＜30岁组8例，30岁～35岁组18例，＞35岁组4例。 2．实验方法：以AOPP作为蛋白氧化指标，采用Vitko-Sarsat等介绍的方法进行测定。 3．统计学方法：所有数据应用spss10.0统计软件进行分析，P＜0.05认为差异有统计学意义。结果以均数±标准差((?)±s)表示，采用偏相关分析，独立样本t检验和One-way ANOVA方差分析。 【结果】 1．常规IVF周期中，第一天胚胎培养液中AOPP浓度在3.2μmol／l以下时，其变化与受精率(r=0.137，P=0.863)、卵裂率(r=0.623，，P=0.377)、碎片10％以下胚胎形成率(r=-0.156，P=0.844)、六细胞以上胚胎形成率(r=-0.444，P=0.556)无相关性；在3.2μmol／l以上时，AOPP水平与受精率(r=-0.226，P=0.338)、卵裂率(r=0.202，P=0.392)、碎片10％以下胚胎形成率(r=-0.232，P=0.325)、六细胞以上胚胎形成率(r=0.008，P=0.975)亦无相关性。 2．ICSI周期中，第一天胚胎培养液中AOPP浓度在3.2μmol／l以下时，其变化与受精率(r=-0.323，P=0.791)、卵裂率(r=0.014，P=0.991)、碎片10％以下胚胎形成率(r=0.238，P=0.847)、六细胞以上胚胎形成率(r=-0.029，P=0.982)无相关性；在3.2μmol／l以上时，AOPP水平与受精率(r=-0.472，P=0.031)、碎片10％以下胚胎形成率(r=-0.482，P=0.027)、六细胞以上胚胎形成率(r=-0.548，P=0.010)呈显著负相关，与卵裂率(r=-0.096，P=0.679)无相关性。 3．常规IVF周期中，非妊娠组中AOPP水平高于妊娠组，有显著性差异(t=3.160，P=0.004)；不同年龄组AOPP水平有显著性差异(F=19.395，P=0.000)，其中不孕妇女年龄＞35岁组AOPP水平最高，＜30岁组最低。 4．ICSI周期中，非妊娠组AOPP水平高于妊娠组，有显著性差异(t=2.421，P=0.022)；不同年龄组AOPP水平有显著性差异(F=3.975，P=0.031)，其中不孕妇女年龄＞35岁组AOPP水平最高，＜30岁组最低。 【结论】 1．IVF-ET周期中，第一天胚胎培养液中存在蛋白氧化应激现象。 2．ICSI周期中，第一天胚胎培养液中高水平的AOPP可能对早期胚胎发育和IVF结局造成不良影响。 3．常规IVF周期中，第一天胚胎培养液中高水平的AOPP虽然不影响早期胚胎发育，但对IVF结局造成不良影响。 第三章低温冷冻和孵育对人精子氧化应激水平的影响 【目的】 探讨低温冷冻和孵育对人精子氧化应激水平的影响。 【方法】 1．标本来源和分组：60例正常人精液标本取自南方医院生殖医学中心因女性因素行试管婴儿治疗的男性患者。将每一例精液标本，梯度离心法处理后，取得沉淀之精子，加人输卵管培养液(human tubal fluid，HTF)，配制成精子密度在(20～30)×10~6／mL的悬液，取2.5 mL均分五份，分为五组，分别进行以下处理： A组为处理前对照组，不进行低温冷冻处理或孵育处理。 B组为低温冷冻空白实验组，去除精子后的精子悬液进行低温冷冻处理。 C组为低温冷冻样本组，含精子的精子悬液进行低温冷冻处理。 D组为孵育空白实验组，去除精子后的精子悬液进行孵育处理。 E组为孵育样本组，含精子的精子悬液进行孵育处理。 2．实验方法：以AOPP作为蛋白氧化指标，采用Vitko-Sarsat等介绍的方法进行测定。以丙二醛(malondialdehyde，MDA)作为脂质过氧化指标，采用硫代巴比妥酸(thiobarbituri，TBA)比色法。 3．统计学方法：所有数据以均数±标准差((?)±s)表示，采用SPSS10.0统计软件进行分析，P＜0.05为差异显著。采用One-way ANOVA方差分析，AOPP水平的多重比较采用Tamhane's T2方法，MDA水平的多重比较采用最小有意义差异(least significant difference，LSD)t检验方法。 【结果】 1．不同处理组之间AOPP水平(F=19.994，P=0.000)和MDA水平(F=31.837，P=0.000)均有显著性差异。 2．低温冷冻样本组MDA水平显著高于处理前对照组(P=0.000)和低温冷冻空白实验组(P=0.000)，但AOPP水平无显著性差异(P_1=0.663；P_2=0.829)。 3．孵育样本组AOPP和MDA水平均显著高于处理前对照组和孵育空白实验组(AOPP：P_1=0.000；P_2=0.000。MDA：P_1=0.000；P_2=0.000)。 4．孵育样本组与低温冷冻样本组比较，AOPP(P=0.000)和MDA(P=0.046)水平均显著升高。 【结论】 1．精子的低温冷冻和孵育，均存在氧化应激损伤。 2．低温冷冻主要造成精子的脂质过氧化损伤，孵育可致精子的脂质过氧化损伤和蛋白氧化损伤。
[Abstract]:With the establishment and development of free radical theory, it has been found that some metabolites of oxygen and their derivatives can damage the body. These substances have more active chemical properties than oxygen. They are collectively called reactive oxygen species (ROS). Under normal conditions, the production and removal of ROS maintain a dynamic balance, but in some pathological conditions, ROS is more active than oxygen. In both cases and senescence, ROS production increases or cellular antioxidant mechanisms are impaired, resulting in the accumulation of ROS, mainly leading to oxidative damage to macromolecules such as deoxyribonucleic acid (DNA), lipids and proteins. DNA oxidative damage can lead to mutations or deletions in nuclear DNA and mitochondrial DNA, and lipid peroxidation in cell membranes leads to the formation of ROS. Membrane fluidity changes, and protein oxidative damage leads to inactivation of important enzymes and impaired protein function.
It was found that ROS was present in follicular fluid, and a certain level of ROS played an important role in maintaining the normal growth of oocytes. Over-elevation of ROS had adverse effects on oocyte maturation and subsequent embryonic development. These results suggest that ROS-induced oxidative stress exists in human follicular fluid, embryonic culture fluid and cryopreserved semen. Previous studies have focused on ROS-mediated lipid peroxidation damage, but recent studies have shown that protein oxygen is present in human follicular fluid, embryonic culture fluid and cryopreserved semen. Chemical modification should not be neglected. However, up to now, there is no report on ROS-mediated protein oxidative damage in human follicular fluid, embryo culture medium and cryopreserved semen. Is there any protein oxidative stress in the above-mentioned system? Is it harmful to the extent? This study intends to study the reproductive effects of ROS and eggs. Based on the study of other human tissues, advanced oxidation protein products (AOPP) were used as an indicator of protein oxidative damage. ROS-mediated protein oxidative damage was observed in human follicular fluid, embryo culture medium and cryopreserved semen, and its relationship with oocytes and early embryos was preliminarily discussed. Developmental relationship.
Chapter 1 Relationship between protein oxidation level in human follicular fluid and IVF-ET outcome parameters
[Objective]
Objective To investigate the relationship between protein oxidation level in follicular fluid and outcome parameters of IVF-ET during in vitro fertilization-embryo transfer (IVF-ET) cycles.
[method]
1. Source and grouping: Follicular fluid specimens were collected from 64 infertile women treated with IVF-ET from July 2005 to April 2006 in the Reproductive Center of Southern Hospital. Case selection criteria: age ranged from 25 to 38 years with an average age of 32.41 (+ 3.17); tubal factor infertility alone; conventional IVF-ET treatment; endometrium on HCG days. The patients were divided into three groups according to the number of eggs harvested: 16 cases in 8 groups, 32 cases in 8-15 groups and 16 cases in 15 groups.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s) or percentage (%). Partial correlation analysis, one-way ANOVA variance analysis and independent sample t test were used.
[results]
1. AOPP levels in follicular fluid were negatively correlated with the ratio 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 formation rate (r = - 0.520, P = 0.000).
2. There were significant differences in AOPP levels among different egg-harvesting groups (F=3.851, P=0.027), among which the lowest AOPP level was found in 8-15 egg-harvesting groups and the highest AOPP level was found in 8 egg-harvesting groups.
3. the level of AOPP in non pregnant group was higher than that in pregnancy group, with significant difference (t=3.665, P=-0.001).
4. There was significant difference in AOPP levels among different age groups (F = 15.919, P = 0.000), and AOPP levels were the highest in the group over 35 years old and the lowest in the group under 30 years old.
[Conclusion]
Oxidative stress is present in follicular fluid during 1.IVF-ET cycle.
2. High levels of AOP in follicular fluid may have adverse effects on oocyte and early embryonic development, and then affect the outcome of IVF.
The second chapter is about the relationship between the level of protein oxidation in embryonic culture medium and the development of early embryos.
[Objective]
Objective to investigate the relationship between protein oxidation level and embryonic development in the first day embryo of IVF-ET cycle.
[method]
1. Sample Sources and Groups: Embryo Culture Medium Samples from the first day of the Southern Hospital Reproduction Center from July to November 2005 routine IVF treatment of 30 patients and intracytoplasmic sperm injection-embryo transplantation (ICSI-ET) treatment of 30 patients. Two groups, 30 cases in the conventional IVF group and 30 cases in the ICSI group. According to the age of infertile women, there were 4 cases in the conventional IVF group, 17 cases in the 30-35-year-old group, 9 cases in the over-35-year-old group, 8 cases in the 30-year-old group, 18 cases in the 30-year-old group and 4 cases in the over-35-year-old group.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All the data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s), partial correlation analysis, independent sample t test and one-way ANOVA analysis of variance.
[results]
1. There was no correlation between AOPP concentration in the first day of IVF cycle and fertilization rate (r = 0.137, P = 0.863), cleavage rate (r = 0.623, P = 0.377), embryo formation rate (r = - 0.156, P = 0.844) and embryo formation rate (r = - 0.444, P = 0.556) above 6 cells in the first day of IVF cycle. There was no correlation between fertilization rate (r = - 0.226, P = 0.338), cleavage rate (r = 0.202, P = 0.392), embryo formation rate below 10% of fragments (r = - 0.232, P = 0.325) and embryo formation rate above six cells (r = 0.008, P = 0.975).
2. During the ICSI cycle, when AOPP concentration in the first day of embryo culture was below 3.2 micromol/l, there was no correlation between AOPP level and fertilization rate (r = - 0.323, P = 0.791), cleavage rate (r = 0.014, P = 0.991), embryo formation rate below 10% of fragments (r = 0.238, P = 0.847), and embryo formation rate above 6 cells (r = - 0.029, P = 0.982). Fertilization rate (r = - 0.472, P = 0.031), embryo formation rate below 10% of fragments (r = - 0.482, P = 0.027), embryo formation rate above six cells (r = - 0.548, P = 0.010) was negatively correlated with cleavage rate (r = - 0.096, P = 0.679).
3. In the conventional IVF cycle, the AOPP level in the non-pregnant group was significantly higher than that in the pregnant group (t = 3.160, P = 0.004); the AOPP level in the different age groups was significantly different (F = 19.395, P = 0.000), and the AOPP level was the highest in the infertile women aged over 35 and the lowest in the women aged under 30.
4. In ICSI cycle, the AOPP level in non-pregnant women was higher than that in pregnant women (t = 2.421, P = 0.022), and there was a significant difference among different age groups (F = 3.975, P = 0.031). The AOPP level in infertile women aged over 35 was the highest, and that in women aged under 30 was the lowest.
[Conclusion]
In the 1.IVF-ET cycle, there was protein oxidative stress on the first day of embryo culture.
2. During the ICSI cycle, high levels of AOP in the first day of embryo culture may have adverse effects on early embryonic development and IVF outcomes.
3. High levels of AOP in the first day of IVF cycle did not affect early embryonic development, but negatively affected the outcome of IVF.
The third chapter is the effect of cryopreservation and incubation on the level of oxidative stress in human sperm.
[Objective]
Objective to investigate the effects of cryopreservation and incubation on oxidative stress in human spermatozoa.
[method]
1. Source and grouping of specimens: 60 normal human semen specimens were taken from male patients treated with in vitro fertilization for female factors in the Reproductive Medical Center of Southern Hospital. The suspension of ~6 / mL was divided into five groups according to 2.5 mL and divided into five groups.
Group A was treated with pre control group without cryopreservation or incubation.
Group B was cryopreserved blank group, and sperm suspension after cryopreservation was cryopreserved.
Group C was cryopreserved, and sperm containing sperm suspension was cryopreserved.
Group D was incubated in blank experimental group, and sperm suspension after spermatozoa was incubated.
Group E was incubated with sperm samples and incubated with sperm suspension.
2. Experimental method: AOPP was used as the index of protein oxidation, and Vitko-Sarsat was used to determine it. Malondialdehyde (MDA) was used as the index of lipid peroxidation, and thiobarbituri (TBA) colorimetry was used.
3. Statistical methods: All data were expressed as mean (?) + standard deviation ((?) + s) and analyzed with SPSS10.0 statistical software, P < 0.05 as significant difference. One-way ANOVA analysis of variance showed that multiple comparisons of AOPP levels were performed with Tamhane's T2 method, and multiple comparisons of MDA levels were performed with least significant difference (LSD). T test method.
[results]
1. There were significant differences in AOPP level (F = 19.994, P = 0.000) and MDA level (F = 31.837, P = 0.000) among different treatment groups.
2. The level of MDA in cryopreservation sample group was significantly higher than that in control group (P = 0.000) and cryopreservation blank group (P = 0.000), but the level of AOPP had no significant difference (P_1 = 0.663; P_2 = 0.829).
3. The levels of AOPP and MDA in incubation sample group were significantly higher than those in control group and incubation blank group before treatment (AOPP: P_1 = 0.000; P_2 = 0.000. MDA: P_1 = 0.000; P_2 = 0.000).
4. the levels of AOPP (P=0.000) and MDA (P=0.046) in the incubated sample group were significantly higher than those in the cryopreservation group.
[Conclusion]
1. there is oxidative stress damage in cryopreservation and incubation of spermatozoa.
2. Cryopreservation mainly causes lipid peroxidation damage of sperm, and incubation can cause lipid peroxidation damage and protein oxidation damage of sperm.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321-33
本文编号：2224845
[Abstract]:With the establishment and development of free radical theory, it has been found that some metabolites of oxygen and their derivatives can damage the body. These substances have more active chemical properties than oxygen. They are collectively called reactive oxygen species (ROS). Under normal conditions, the production and removal of ROS maintain a dynamic balance, but in some pathological conditions, ROS is more active than oxygen. In both cases and senescence, ROS production increases or cellular antioxidant mechanisms are impaired, resulting in the accumulation of ROS, mainly leading to oxidative damage to macromolecules such as deoxyribonucleic acid (DNA), lipids and proteins. DNA oxidative damage can lead to mutations or deletions in nuclear DNA and mitochondrial DNA, and lipid peroxidation in cell membranes leads to the formation of ROS. Membrane fluidity changes, and protein oxidative damage leads to inactivation of important enzymes and impaired protein function.
It was found that ROS was present in follicular fluid, and a certain level of ROS played an important role in maintaining the normal growth of oocytes. Over-elevation of ROS had adverse effects on oocyte maturation and subsequent embryonic development. These results suggest that ROS-induced oxidative stress exists in human follicular fluid, embryonic culture fluid and cryopreserved semen. Previous studies have focused on ROS-mediated lipid peroxidation damage, but recent studies have shown that protein oxygen is present in human follicular fluid, embryonic culture fluid and cryopreserved semen. Chemical modification should not be neglected. However, up to now, there is no report on ROS-mediated protein oxidative damage in human follicular fluid, embryo culture medium and cryopreserved semen. Is there any protein oxidative stress in the above-mentioned system? Is it harmful to the extent? This study intends to study the reproductive effects of ROS and eggs. Based on the study of other human tissues, advanced oxidation protein products (AOPP) were used as an indicator of protein oxidative damage. ROS-mediated protein oxidative damage was observed in human follicular fluid, embryo culture medium and cryopreserved semen, and its relationship with oocytes and early embryos was preliminarily discussed. Developmental relationship.
Chapter 1 Relationship between protein oxidation level in human follicular fluid and IVF-ET outcome parameters
[Objective]
Objective To investigate the relationship between protein oxidation level in follicular fluid and outcome parameters of IVF-ET during in vitro fertilization-embryo transfer (IVF-ET) cycles.
[method]
1. Source and grouping: Follicular fluid specimens were collected from 64 infertile women treated with IVF-ET from July 2005 to April 2006 in the Reproductive Center of Southern Hospital. Case selection criteria: age ranged from 25 to 38 years with an average age of 32.41 (+ 3.17); tubal factor infertility alone; conventional IVF-ET treatment; endometrium on HCG days. The patients were divided into three groups according to the number of eggs harvested: 16 cases in 8 groups, 32 cases in 8-15 groups and 16 cases in 15 groups.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s) or percentage (%). Partial correlation analysis, one-way ANOVA variance analysis and independent sample t test were used.
[results]
1. AOPP levels in follicular fluid were negatively correlated with the ratio 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 formation rate (r = - 0.520, P = 0.000).
2. There were significant differences in AOPP levels among different egg-harvesting groups (F=3.851, P=0.027), among which the lowest AOPP level was found in 8-15 egg-harvesting groups and the highest AOPP level was found in 8 egg-harvesting groups.
3. the level of AOPP in non pregnant group was higher than that in pregnancy group, with significant difference (t=3.665, P=-0.001).
4. There was significant difference in AOPP levels among different age groups (F = 15.919, P = 0.000), and AOPP levels were the highest in the group over 35 years old and the lowest in the group under 30 years old.
[Conclusion]
Oxidative stress is present in follicular fluid during 1.IVF-ET cycle.
2. High levels of AOP in follicular fluid may have adverse effects on oocyte and early embryonic development, and then affect the outcome of IVF.
The second chapter is about the relationship between the level of protein oxidation in embryonic culture medium and the development of early embryos.
[Objective]
Objective to investigate the relationship between protein oxidation level and embryonic development in the first day embryo of IVF-ET cycle.
[method]
1. Sample Sources and Groups: Embryo Culture Medium Samples from the first day of the Southern Hospital Reproduction Center from July to November 2005 routine IVF treatment of 30 patients and intracytoplasmic sperm injection-embryo transplantation (ICSI-ET) treatment of 30 patients. Two groups, 30 cases in the conventional IVF group and 30 cases in the ICSI group. According to the age of infertile women, there were 4 cases in the conventional IVF group, 17 cases in the 30-35-year-old group, 9 cases in the over-35-year-old group, 8 cases in the 30-year-old group, 18 cases in the 30-year-old group and 4 cases in the over-35-year-old group.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All the data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s), partial correlation analysis, independent sample t test and one-way ANOVA analysis of variance.
[results]
1. There was no correlation between AOPP concentration in the first day of IVF cycle and fertilization rate (r = 0.137, P = 0.863), cleavage rate (r = 0.623, P = 0.377), embryo formation rate (r = - 0.156, P = 0.844) and embryo formation rate (r = - 0.444, P = 0.556) above 6 cells in the first day of IVF cycle. There was no correlation between fertilization rate (r = - 0.226, P = 0.338), cleavage rate (r = 0.202, P = 0.392), embryo formation rate below 10% of fragments (r = - 0.232, P = 0.325) and embryo formation rate above six cells (r = 0.008, P = 0.975).
2. During the ICSI cycle, when AOPP concentration in the first day of embryo culture was below 3.2 micromol/l, there was no correlation between AOPP level and fertilization rate (r = - 0.323, P = 0.791), cleavage rate (r = 0.014, P = 0.991), embryo formation rate below 10% of fragments (r = 0.238, P = 0.847), and embryo formation rate above 6 cells (r = - 0.029, P = 0.982). Fertilization rate (r = - 0.472, P = 0.031), embryo formation rate below 10% of fragments (r = - 0.482, P = 0.027), embryo formation rate above six cells (r = - 0.548, P = 0.010) was negatively correlated with cleavage rate (r = - 0.096, P = 0.679).
3. In the conventional IVF cycle, the AOPP level in the non-pregnant group was significantly higher than that in the pregnant group (t = 3.160, P = 0.004); the AOPP level in the different age groups was significantly different (F = 19.395, P = 0.000), and the AOPP level was the highest in the infertile women aged over 35 and the lowest in the women aged under 30.
4. In ICSI cycle, the AOPP level in non-pregnant women was higher than that in pregnant women (t = 2.421, P = 0.022), and there was a significant difference among different age groups (F = 3.975, P = 0.031). The AOPP level in infertile women aged over 35 was the highest, and that in women aged under 30 was the lowest.
[Conclusion]
In the 1.IVF-ET cycle, there was protein oxidative stress on the first day of embryo culture.
2. During the ICSI cycle, high levels of AOP in the first day of embryo culture may have adverse effects on early embryonic development and IVF outcomes.
3. High levels of AOP in the first day of IVF cycle did not affect early embryonic development, but negatively affected the outcome of IVF.
The third chapter is the effect of cryopreservation and incubation on the level of oxidative stress in human sperm.
[Objective]
Objective to investigate the effects of cryopreservation and incubation on oxidative stress in human spermatozoa.
[method]
1. Source and grouping of specimens: 60 normal human semen specimens were taken from male patients treated with in vitro fertilization for female factors in the Reproductive Medical Center of Southern Hospital. The suspension of ~6 / mL was divided into five groups according to 2.5 mL and divided into five groups.
Group A was treated with pre control group without cryopreservation or incubation.
Group B was cryopreserved blank group, and sperm suspension after cryopreservation was cryopreserved.
Group C was cryopreserved, and sperm containing sperm suspension was cryopreserved.
Group D was incubated in blank experimental group, and sperm suspension after spermatozoa was incubated.
Group E was incubated with sperm samples and incubated with sperm suspension.
2. Experimental method: AOPP was used as the index of protein oxidation, and Vitko-Sarsat was used to determine it. Malondialdehyde (MDA) was used as the index of lipid peroxidation, and thiobarbituri (TBA) colorimetry was used.
3. Statistical methods: All data were expressed as mean (?) + standard deviation ((?) + s) and analyzed with SPSS10.0 statistical software, P < 0.05 as significant difference. One-way ANOVA analysis of variance showed that multiple comparisons of AOPP levels were performed with Tamhane's T2 method, and multiple comparisons of MDA levels were performed with least significant difference (LSD). T test method.
[results]
1. There were significant differences in AOPP level (F = 19.994, P = 0.000) and MDA level (F = 31.837, P = 0.000) among different treatment groups.
2. The level of MDA in cryopreservation sample group was significantly higher than that in control group (P = 0.000) and cryopreservation blank group (P = 0.000), but the level of AOPP had no significant difference (P_1 = 0.663; P_2 = 0.829).
3. The levels of AOPP and MDA in incubation sample group were significantly higher than those in control group and incubation blank group before treatment (AOPP: P_1 = 0.000; P_2 = 0.000. MDA: P_1 = 0.000; P_2 = 0.000).
4. the levels of AOPP (P=0.000) and MDA (P=0.046) in the incubated sample group were significantly higher than those in the cryopreservation group.
[Conclusion]
1. there is oxidative stress damage in cryopreservation and incubation of spermatozoa.
2. Cryopreservation mainly causes lipid peroxidation damage of sperm, and incubation can cause lipid peroxidation damage and protein oxidation damage of sperm.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321-33
【参考文献】
相关期刊论文 前1条
1 余月明,侯凡凡,周华,杨燕,张训,杨凌,胡敏燕;慢性肾衰竭患者同型半胱氨酸血症与动脉粥样硬化的关系[J];中华内科杂志;2002年08期
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