采用偏振光显微镜分析透明带在受精过程中的作用
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摘要
第一部分采用偏振光显微镜对受精前后及异常受精卵透明带的观察研究
目的
采用偏振光显微镜(polarized light microscope, Polscope)观察并测量人卵子及合子透明带密度(zona pellucida density, ZPD)、透明带厚度(zona pellucida thickness, ZPT),分析体外成熟过程中透明带(zona pellucida, ZP)的变化及受精后ZP的变化、不同授精方式受精后合子ZP的差别、及相同体外授精方式(in vitro fertilization, IVF)中ZP与不同受精结局的相关性。
方法
IVF授精后18-20 h采用偏振光显微镜对68例中248枚受精卵和60枚未成熟卵子摄像;单精子胞浆内显微注射(Intracytoplasmic sperm injection, ICSI)授精后18-20 h对11例中61枚受精卵子摄像;另外36个ICSI周期127枚卵子,卵子取出2-4 h后拆除颗粒细胞,摄像。软件测量所拍摄图像的ZP,分析成熟过程中及受精后ZP的变化、不同受精方式ZP的差别及不同受精结局ZP的差别。
结果
未成熟卵全层ZPT、内层ZPD和ZPT高于成熟卵子,IVF受精后各层ZPT、ZPD显著增大(2.49±0.49, 8.79±1.91, 19.33±2.28 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05);IVF和ICSI授精后受精卵ZP有显著差别;IVF中3原核(3 pronuclear, 3PN)受精卵内层ZPT低于2PN(8.19±1.68 vs. 8.79±1.91; p<0.05),0PN全层及内层ZPT、ZPD均高于2PN(2.81±0.69, 9.55±1.45, 20.79±3.41 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05)。
结论
成熟度不同卵子密度和厚度不同,卵子受精后ZPT、ZPD发生变化;不同授精方式ZP存在差别;受精卵ZP结构与受精结局有相关性。
第二部分6h短时授精与18h隔夜授精透明带变化的随机自身对照研究
目的
6 h与18 h精卵共培养组间受精卵ZP和胚胎质量的关系
方法
21周期258枚卵子随机分6 h和18 h精卵共培养组自身对照,受精后20 h偏振光显微镜摄像,分析两组间正常受精卵ZP和胚胎质量的差异。
结果
6 h与18 h组比较,受精率及多精受精率没有差异,可移植胚胎率略高(70.1% vs. 63.27%)无统计差异,移植胚胎所占比率高(67.80 vs. 37.21%)。6 h组中正常受精卵内层、全层透明带厚度(zona pellucida thickness, ZPT),内层透明带密度(zona pellucida density, ZPD)(8.28±1.28, 18.97±2.64, 2.09±0.50vs. 8.86±1.52, 19.57±2.66, 2.24±0.67)低于18 h组;发育为可移植胚胎的受精卵ZPD (2.07±0.49 vs. 2.26±0.72)显著低于18 h组,而发育为不可移植胚胎的受精卵外层ZPT (9.14±2.46 vs. 7.74±1.37)高于18 h组。
结论
短时受精组合子透明带内层厚度、密度较传统隔夜受精组合子低,这可能有利于胚胎孵出。
PART ONE ANALYSING ZONA PELLUCIDA OF ZYGOTE BY POLSCOPE AFTER FERTILIZATION
Objective
In human in-vitro fertilization (IVF), the failure and low fertilization rate is about 10%~30% and the abnormal fertilization rate is about 2%~9%. Oocyte quality plays an important role in fertilization. Zona pellucide (ZP), the transparent extracellular matrix, the way where sperm must penetrate thereafter form a barrier arrested more sperm penetrate. The characteristic of ZP may be related to fertilization outcome. The polarized light microscope (Polscope) was used in this study to investigate the difference of zona pellucida between germinal vesicle present (GV), metaphase I (MI), approaching metaphase II (AMII), metaphase II (MII); two-pronuclear (2PN) from intracytoplasmic sperm injection (ICSI) and 2PN from IVF; 2PN, three- pronuclear (3PN), and zero-pronuclear (0PN) from IVF.
Methods
The zona pellucida of 127 good quality matured oocytes, 80 unmatured oocytes, 69 2PN zygotes, 26 0PN zygotes, 93 3PN zygotes from IVF and 61 2PN zygotes from ICSI were imaged by Polscope.
Results
ZPT and ZPD of inner layer (IL) and ZPT of total layer (TL) of oocyte after full grown gradually decreased until MII. The ZPT and ZPD of IL and ZPT of the TL of 2PN derived from IVF was higher than MII (2.49±0.49, 8.79±1.91, 19.33±2.28; 2.36±0.65, 8.80±1.54, 19.37±2.38 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05) ZPT of 2PN derived from IVF was higher than 2PN derived from ICSI too. In IVF the ZPT and ZPD of IL and ZPT of the TL of 0PN zygotes were higher than 2PN (2.49±0.49, 8.79±1.91, 19.33±2.28 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05), whereas the IL ZPT of 3PN zygotes was lower than 2PN (8.19±1.68 vs. 8.79±1.91; p<0.05).
Conclusions
After oocyte full grown, ZP was gradually thinner and looser until MII. After ZP reaction, the ZP structure changed. There are different between IVF and ICSI. The status of ZP was the cause of abnormal fertilization even when the sperm were considered normal.
PART TWO CHANGE OF ZONA PELLUCIDA BETWEEN 6H SHORTEN GAMETS CO-INCUBATION AND 18H GAMETS CO-INCUBATION?
Objective
The positive value of the short gametes co-incubations on embryo quality is still controversial. A study showed that short gametes co-incubations reduced zone pellucida thickness (ZPT) which was evidence of better culture condition. To further investigate these finding, sibling oocytes (n=258) from 21 IVF couples were randomized allocated to a 6 h or 18 h co-incubations group to compare zone pellucida (ZP) and embryo quality (EQ).
Method
Oocytes (n=258) from 21 IVF couples were randomly allocated to 6 h group or 18 h group. Mean ZPT and ZP density (ZPD) of zygote were measured by polarized light microscope (Polscope) 20 h after fertilization. Other targets were normal fertilization (two-pronuclear, 2PN), EQ, clinical pregnancy (CP) and implantation (IR).
Result
Rate of normal fertilization and 3PN and proportion of high quality embryo were not statistically different between two groups. However, ZPD and ZPT of inner layer and ZPT of full layers for 2PN zygote were significantly thinner in 6 h than 18 h of incubations (2.09±0.50, 8.28±1.28, 18.97±2.64 vs. 2.24±0.67 8.86±1.52 19.57±2.66; P<0.05). ZPD of inner layer for grade 1-3 embryo was also lower from 6 h incubation. In contrast, ZPT of outer layer for grade 4 embryos was thicker at 6 h incubation. Overall, ZPT of inner layer was negatively related to embryo quality.
Conclusions
Short gametes co-incubations reduced ZPT and ZPD of zygote which may be benefit to embryo hatching.
目的
采用偏振光显微镜(polarized light microscope, Polscope)观察并测量人卵子及合子透明带密度(zona pellucida density, ZPD)、透明带厚度(zona pellucida thickness, ZPT),分析体外成熟过程中透明带(zona pellucida, ZP)的变化及受精后ZP的变化、不同授精方式受精后合子ZP的差别、及相同体外授精方式(in vitro fertilization, IVF)中ZP与不同受精结局的相关性。
方法
IVF授精后18-20 h采用偏振光显微镜对68例中248枚受精卵和60枚未成熟卵子摄像;单精子胞浆内显微注射(Intracytoplasmic sperm injection, ICSI)授精后18-20 h对11例中61枚受精卵子摄像;另外36个ICSI周期127枚卵子,卵子取出2-4 h后拆除颗粒细胞,摄像。软件测量所拍摄图像的ZP,分析成熟过程中及受精后ZP的变化、不同受精方式ZP的差别及不同受精结局ZP的差别。
结果
未成熟卵全层ZPT、内层ZPD和ZPT高于成熟卵子,IVF受精后各层ZPT、ZPD显著增大(2.49±0.49, 8.79±1.91, 19.33±2.28 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05);IVF和ICSI授精后受精卵ZP有显著差别;IVF中3原核(3 pronuclear, 3PN)受精卵内层ZPT低于2PN(8.19±1.68 vs. 8.79±1.91; p<0.05),0PN全层及内层ZPT、ZPD均高于2PN(2.81±0.69, 9.55±1.45, 20.79±3.41 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05)。
结论
成熟度不同卵子密度和厚度不同,卵子受精后ZPT、ZPD发生变化;不同授精方式ZP存在差别;受精卵ZP结构与受精结局有相关性。
第二部分6h短时授精与18h隔夜授精透明带变化的随机自身对照研究
目的
6 h与18 h精卵共培养组间受精卵ZP和胚胎质量的关系
方法
21周期258枚卵子随机分6 h和18 h精卵共培养组自身对照,受精后20 h偏振光显微镜摄像,分析两组间正常受精卵ZP和胚胎质量的差异。
结果
6 h与18 h组比较,受精率及多精受精率没有差异,可移植胚胎率略高(70.1% vs. 63.27%)无统计差异,移植胚胎所占比率高(67.80 vs. 37.21%)。6 h组中正常受精卵内层、全层透明带厚度(zona pellucida thickness, ZPT),内层透明带密度(zona pellucida density, ZPD)(8.28±1.28, 18.97±2.64, 2.09±0.50vs. 8.86±1.52, 19.57±2.66, 2.24±0.67)低于18 h组;发育为可移植胚胎的受精卵ZPD (2.07±0.49 vs. 2.26±0.72)显著低于18 h组,而发育为不可移植胚胎的受精卵外层ZPT (9.14±2.46 vs. 7.74±1.37)高于18 h组。
结论
短时受精组合子透明带内层厚度、密度较传统隔夜受精组合子低,这可能有利于胚胎孵出。
PART ONE ANALYSING ZONA PELLUCIDA OF ZYGOTE BY POLSCOPE AFTER FERTILIZATION
Objective
In human in-vitro fertilization (IVF), the failure and low fertilization rate is about 10%~30% and the abnormal fertilization rate is about 2%~9%. Oocyte quality plays an important role in fertilization. Zona pellucide (ZP), the transparent extracellular matrix, the way where sperm must penetrate thereafter form a barrier arrested more sperm penetrate. The characteristic of ZP may be related to fertilization outcome. The polarized light microscope (Polscope) was used in this study to investigate the difference of zona pellucida between germinal vesicle present (GV), metaphase I (MI), approaching metaphase II (AMII), metaphase II (MII); two-pronuclear (2PN) from intracytoplasmic sperm injection (ICSI) and 2PN from IVF; 2PN, three- pronuclear (3PN), and zero-pronuclear (0PN) from IVF.
Methods
The zona pellucida of 127 good quality matured oocytes, 80 unmatured oocytes, 69 2PN zygotes, 26 0PN zygotes, 93 3PN zygotes from IVF and 61 2PN zygotes from ICSI were imaged by Polscope.
Results
ZPT and ZPD of inner layer (IL) and ZPT of total layer (TL) of oocyte after full grown gradually decreased until MII. The ZPT and ZPD of IL and ZPT of the TL of 2PN derived from IVF was higher than MII (2.49±0.49, 8.79±1.91, 19.33±2.28; 2.36±0.65, 8.80±1.54, 19.37±2.38 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05) ZPT of 2PN derived from IVF was higher than 2PN derived from ICSI too. In IVF the ZPT and ZPD of IL and ZPT of the TL of 0PN zygotes were higher than 2PN (2.49±0.49, 8.79±1.91, 19.33±2.28 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05), whereas the IL ZPT of 3PN zygotes was lower than 2PN (8.19±1.68 vs. 8.79±1.91; p<0.05).
Conclusions
After oocyte full grown, ZP was gradually thinner and looser until MII. After ZP reaction, the ZP structure changed. There are different between IVF and ICSI. The status of ZP was the cause of abnormal fertilization even when the sperm were considered normal.
PART TWO CHANGE OF ZONA PELLUCIDA BETWEEN 6H SHORTEN GAMETS CO-INCUBATION AND 18H GAMETS CO-INCUBATION?
Objective
The positive value of the short gametes co-incubations on embryo quality is still controversial. A study showed that short gametes co-incubations reduced zone pellucida thickness (ZPT) which was evidence of better culture condition. To further investigate these finding, sibling oocytes (n=258) from 21 IVF couples were randomized allocated to a 6 h or 18 h co-incubations group to compare zone pellucida (ZP) and embryo quality (EQ).
Method
Oocytes (n=258) from 21 IVF couples were randomly allocated to 6 h group or 18 h group. Mean ZPT and ZP density (ZPD) of zygote were measured by polarized light microscope (Polscope) 20 h after fertilization. Other targets were normal fertilization (two-pronuclear, 2PN), EQ, clinical pregnancy (CP) and implantation (IR).
Result
Rate of normal fertilization and 3PN and proportion of high quality embryo were not statistically different between two groups. However, ZPD and ZPT of inner layer and ZPT of full layers for 2PN zygote were significantly thinner in 6 h than 18 h of incubations (2.09±0.50, 8.28±1.28, 18.97±2.64 vs. 2.24±0.67 8.86±1.52 19.57±2.66; P<0.05). ZPD of inner layer for grade 1-3 embryo was also lower from 6 h incubation. In contrast, ZPT of outer layer for grade 4 embryos was thicker at 6 h incubation. Overall, ZPT of inner layer was negatively related to embryo quality.
Conclusions
Short gametes co-incubations reduced ZPT and ZPD of zygote which may be benefit to embryo hatching.
引文
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