输卵管积水对子宫内膜容受性影响的研究
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摘要
输卵管积水对子宫内膜容受性影响的研究
目的:研究输卵管积水对IVF-ET结局的影响;探讨输卵管积水对窗口期子宫内膜组织基因表达谱的改变,筛选出输卵管积水影响子宫内膜容受性的差异基因;并对其中的差异表达基因在子宫内膜的mRNA及蛋白表达水平进行验证,探讨其临床意义。
方法:
1.统计分析:2003年8月～2005年8月输卵管积水行IVF-ET 143个周期(积水组)及输卵管其它病变无积水行IVF-ET 263个周期(阻塞组),比较两组IVF-ET结局的差异。
2.寡核苷酸芯片技术:取输卵管积水、输卵管阻塞不伴积水和正常女性窗口期子宫内膜组织,分别提取组织总RNA、逆转录成cDNA、生物素标记合成cRNA探针,与Affymetrix U133A2.0基因表达谱芯片杂交,用Affymetrix公司的GCOS1.2软件进行数据分析,以SignalLog Ratio≧2或≦-2的数据点为显著性差异表达基因的筛选标准,通过网上数据库查询基因的功能,进行差异表达基因功能分类。
3.real-time PCR实验:从上述筛选出的差异表达基因谱中,选择有显著差异表达的骨桥蛋白(OPN)和GdA(又名胎盘蛋白14,PP14)以及与OPN相互结合构成胚胎粘附的整合素β3作为研究指标。采用real-time PCR方法对31例输卵管积水、29例输卵管阻塞不伴积水以及21例正常对照患者窗口期子宫内膜组织中上述三种基因表达量进行验证。
4.免疫组织化学实验:对各组患者子宫内膜中骨桥蛋白(OPN)、整合素β3和GdA相应蛋白表达变化进行免疫组织化学分析验证。
SPSS13.0统计软件包进行分析。P＜0.05作为检验水准。
结果:
1.IVF-ET治疗结局
在移植同等质量的胚胎后,输卵管积水组的临床妊娠率32.87%、胚胎着床率18.61%,早期流产率19.15%、异位妊娠率17.02%;输卵管阻塞组临床妊娠率49.8%、胚胎着床率31.87%,早期流产率6.87%、异位妊娠率4.58%。
2.DNA微阵列
(1)差异表达基因积水组比正常组、阻塞组比正常组、积水组比阻塞组,分别筛选出显著差异表达基因588条、180条和750条,按基因主要功能可分为:酶、细胞粘附分子、细胞骨架蛋白相关基因、细胞周期调节因子、金属离子结合蛋白、信号转导蛋白相关基因、免疫反应相关蛋白、载体蛋白相关基因、功能未知的基因等。
(2)积水组比正常组表达差异基因共有588条基因表达有差异,其中表达上调为351条,表达下调为237条。表达上调的和表达下调基因中均以酶活性相关基因最多、其次是信号转导蛋白基因。
(3)阻塞组比正常组表达差异基因共有180条基因表达有差异,其中表达上调为106条,表达下调为74条。表达差异基因亦以酶活性相关基因最多、其次是信号转导蛋白基因。
(4)积水组比阻塞组表达差异基因共有750条基因表达有差异,其中表达上调为302条,表达下调为448条。表达差异基因除外酶活性相关基因最多的是信号转导蛋白基因。
3.Real-time PCR实验:
(1)三组患者窗口期子宫内膜组织中OPN基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中OPN相对表达量依次为0.6238±0.2690、0.8955±0.3887、和1.1065±0.7821;三组间两两相比OPN均无明显差异。
(2)三组患者窗口期子宫内膜组织中整合素β3基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中整合素β3相对表达量依次为0.1945±0.1024、0.8285±0.1945和1.2541±0.5245;积水组与阻塞组相比,积水组与对照组相比有明显差异。
(3)三组患者窗口期子宫内膜组织中GdA基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中GdA相对表达量依次为
2.0815±0.3425、3.1578±0.6712、和4.1257±0.9476;积水组与对照组相比、阻塞组与对照组相比有明显差异。
4.免疫组织化学实验:
(1)三组患者窗口期子宫内膜组织中OPN的表达差异OPN定位于子宫内膜腺上皮及腔上皮细胞,基质细胞中无表达或仅极少量表达。三组相比无显著性差异。
(2)三组患者窗口期子宫内膜组织中整合素β3的表达差异整合素β3定位于子宫内膜腺上皮细胞和(或)腔上皮细胞,基质细胞仅极少量表达。大部分的输卵管积水患者的子宫内膜上整合素β3表达缺失或呈弱阳性。积水组的表达强度显著低于阻塞组及对照组。
(3)三组患者窗口期子宫内膜组织中GdA的表达差异GdA定位于子宫内膜腺上皮及腔上皮细胞,基质细胞中无表达或仅极少量表达。积水组和阻塞组的表达强度较对照组都明显降低。
结论:
1.输卵管积水患者IVF-ET治疗的临床妊娠率低、早期流产率高;
2.输卵管积水对窗口期子宫内膜多种基因的表达产生影响;其中表达差异最大的基因是酶活性相关基因和信号转导蛋白基因。
3.输卵管积水和输卵管其他病变不影响窗口期子宫内膜OPN基因的表达、转录及其蛋白质的合成分泌;
4.输卵管积水抑制窗口期子宫内膜上整合素β3基因表达、转录及其蛋白合成分泌,降低子宫内膜容受胚胎粘附、着床能力;
5.输卵管积水降低窗口期子宫内膜上GdA基因表达、转录及其蛋白质合成分泌,破坏子宫内膜对胚胎的免疫耐受;
Objective: To study the impaction of hydrosapinx on the outcome of IVF-ET. To explore gene expression changes of hydrosapinx patient in the window of implantation (WOI), demonstrate differentially expressed genes, and explore their clinical significance. Validation of array data was accomplished by mRNA quantification by real time quantitative fluorescent PCR (RT-PCR) and by level of protein expression of some differentially expressed genes. And explore their clinical meaning.
Methods: Statistic Analysis: Two groups composed of patients between August 2003 and August 2005 are built. Hydrosapix group is patients with hydrosapinx treated with IVF-ET for 143 cycles. Tubal obstruction group is patients with other pathologic change without hydrosapinx treated with IVF-ET for 263 cycles. Compare the two groups' IVF-ET outcome.
Oligonucleotide Microarray : The differentially expressed genes (Signal Log Ratio (?)2 or (?)2) of endometrium were analyzed among hydrosapinx, tubal obstruction without hydrospinx and healthy volunteer in WOI, using Affymetrix U133A 2.0 gene chip, Functional classification of differentially expressed genes were established by searching in the human data-base.
RT-PCR : Osteopontin (OPN or SPPI), Placenta protein-14 (PP-14, Glycodelin, GdA or PAEP) and Integrinβ_3, which is differentially expressed or closely related with endometrium receptivity, were verified by PT-PCR. The mRNA quantification of 31 hydrosapinx patients, 29 tubal obstruction patients without hydrosapinx and 21 healthy volunteers were analyzed.
Immunohistochemistry: Analyze and verify the changes of three groups' OPN, GdA and Integrinβ_3 by Immunohistochemistry.
SPSS 13.0 statistic package was used and p<0.05 was adopted as significance level.
Results:
1. The treatment outcome of IVE-ET
The quality of transferred embryo between hydrosapinx and tubal obstruction group has no statistical difference. In the hydrosapinx group, the rates of clinical pregnancy, embryo implantation, early abortion and ectopic pregnancy were 32.87%, 18.61%, 19.15% and 17.02%. In the bubal obstruction group, the rates of clinical pregnancy, embryo implantation, early abortion and ectopic pregnancy were 49.8%, 31.87%, 6.87% and 4.58%.
2. Oligonucleotide Microarray:
(1) The differentially expressed genes
There were 588 genes significantly differentially expressed between hydrosapinx patient and healthy volunteer, 180 genes between tubal obstruction patient and healthy volunteer, 750 genes between hydrosapinx and tubal obstruction patient without hydrosapinx. Those genes were classified into 10 groups according to their main function. They are enzyme, cell adhesion, structural protein, regulation of cell cycle, ion binding protein, signal-transporting proteins, immunomodulators, carrier proteins, unknown function ect.
(2) Differentially expressed genes between hydrosapinx patient and healthy volunteer
Total of 588 genes were significantly differentially expressed. 351 genes were up-regulated, and 237 genes were down-regulated. Enzyme related genes were at most among both up-regulated and down-regulated genes. Signal-transporting proteins were second.
(3) Differentially expressed genes between tubal obstruction patient without hydrosapinx and healthy volunteer
Total of 180 genes were significantly differentially expressed. 106 genes were up-regulated, and 74 genes were down-regulated. Enzyme related genes were at most among significantly differentially expressed genes. Signal-transporting proteins were next.
(4) Differentially expressed genes between hydrosapinx and tubal obstruction patient
Total of 750 genes were significantly differentially expressed. 302 genes were up-regulated, and 448 genes were down-regulated. Signal-transporting proteins were the most among significantly differentially expressed genes except Enzyme.
3.RT-PCR
(1) The different expression of OPN genes mRNA among three groups
The OPN relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 0.6238±0.2690, 0.8955±0.3887 and 1.1065±0.7821. There was no obvious difference between any two groups.
(2) The different expression of Integrinβ_3 genes mRNA among three groups
The Integrinβ_3 relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 0.1945±0.1024, 0.8285±0.1945 and 1.2541±0.5245.There was obvious difference between hydrosapinx group and tubal obstruction group. So was hydrosapinx group and health control group.
(3) The different expression of GdA genes mRNA among three groups
The GdA relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 2.0815±0.3425, 3.1578±0.6712 and 4.1257±0.9476. There was obvious difference between hydrosapinx group and health control group. So was hydrosapinx group and health control group.
4. Immunohistochemistry
(1) The different expression of OPN among three groups
OPN was situated in endometrium glandular epithelium and cavitas epithelial cells. There was little expression in stroma-cell. There was no significant difference among three groups.
(2) The different expression of Integrinβ_3 among three groups
Integrinβ_3 was situated in endometrium glandular epithelium and (or) cavitas epithelial cells. There was only a little expression in stroma-cell. The expression of Integrinβ_3 in most hydrosapinx patients' endometrium is absent or weakly positive. The intension of expression in hydrosapinx group is obviously lower than tubal obstruction group and healthy control group.
(3) The different expression of GdA among three groups
GdA was situated in endometrium glandular epithelium and (or) cavitas epithelial cells. There was little expression in stroma-cell. Both hydrosapinx group and tubal obstruction group are obviously lower than healthy control group.
Conclusion:
1. Hydrosapinx patient treated with IVF-ET has low clinical pregnancy rate, while high early abortion rate.
2. Hydrosapinx has influence on many endometrium genes' expression. The most differentially expressed genes are enzyme related genes and signal-transporting protein genes.
3. Hydrosapinx has no effects on OPN gene expression, transcription, protein synthesis and secretion in WOI endometrium. So does tubal obstruction
4. Hydrosapinx inhibits Integrinβ_3 gene expression, transcription, protein synthesis and secretion in WOI endometrium. So that destroys the ability of endometrium recepting embryo adhesion and implantation.
6. Hydrosapinx inhibits GdA gene expression, transcription, proteinsynthesis and secretion in WOI endometrium. And destroys the immune tolerance of endometrium to embryo.
目的:研究输卵管积水对IVF-ET结局的影响;探讨输卵管积水对窗口期子宫内膜组织基因表达谱的改变,筛选出输卵管积水影响子宫内膜容受性的差异基因;并对其中的差异表达基因在子宫内膜的mRNA及蛋白表达水平进行验证,探讨其临床意义。
方法:
1.统计分析:2003年8月～2005年8月输卵管积水行IVF-ET 143个周期(积水组)及输卵管其它病变无积水行IVF-ET 263个周期(阻塞组),比较两组IVF-ET结局的差异。
2.寡核苷酸芯片技术:取输卵管积水、输卵管阻塞不伴积水和正常女性窗口期子宫内膜组织,分别提取组织总RNA、逆转录成cDNA、生物素标记合成cRNA探针,与Affymetrix U133A2.0基因表达谱芯片杂交,用Affymetrix公司的GCOS1.2软件进行数据分析,以SignalLog Ratio≧2或≦-2的数据点为显著性差异表达基因的筛选标准,通过网上数据库查询基因的功能,进行差异表达基因功能分类。
3.real-time PCR实验:从上述筛选出的差异表达基因谱中,选择有显著差异表达的骨桥蛋白(OPN)和GdA(又名胎盘蛋白14,PP14)以及与OPN相互结合构成胚胎粘附的整合素β3作为研究指标。采用real-time PCR方法对31例输卵管积水、29例输卵管阻塞不伴积水以及21例正常对照患者窗口期子宫内膜组织中上述三种基因表达量进行验证。
4.免疫组织化学实验:对各组患者子宫内膜中骨桥蛋白(OPN)、整合素β3和GdA相应蛋白表达变化进行免疫组织化学分析验证。
SPSS13.0统计软件包进行分析。P＜0.05作为检验水准。
结果:
1.IVF-ET治疗结局
在移植同等质量的胚胎后,输卵管积水组的临床妊娠率32.87%、胚胎着床率18.61%,早期流产率19.15%、异位妊娠率17.02%;输卵管阻塞组临床妊娠率49.8%、胚胎着床率31.87%,早期流产率6.87%、异位妊娠率4.58%。
2.DNA微阵列
(1)差异表达基因积水组比正常组、阻塞组比正常组、积水组比阻塞组,分别筛选出显著差异表达基因588条、180条和750条,按基因主要功能可分为:酶、细胞粘附分子、细胞骨架蛋白相关基因、细胞周期调节因子、金属离子结合蛋白、信号转导蛋白相关基因、免疫反应相关蛋白、载体蛋白相关基因、功能未知的基因等。
(2)积水组比正常组表达差异基因共有588条基因表达有差异,其中表达上调为351条,表达下调为237条。表达上调的和表达下调基因中均以酶活性相关基因最多、其次是信号转导蛋白基因。
(3)阻塞组比正常组表达差异基因共有180条基因表达有差异,其中表达上调为106条,表达下调为74条。表达差异基因亦以酶活性相关基因最多、其次是信号转导蛋白基因。
(4)积水组比阻塞组表达差异基因共有750条基因表达有差异,其中表达上调为302条,表达下调为448条。表达差异基因除外酶活性相关基因最多的是信号转导蛋白基因。
3.Real-time PCR实验:
(1)三组患者窗口期子宫内膜组织中OPN基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中OPN相对表达量依次为0.6238±0.2690、0.8955±0.3887、和1.1065±0.7821;三组间两两相比OPN均无明显差异。
(2)三组患者窗口期子宫内膜组织中整合素β3基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中整合素β3相对表达量依次为0.1945±0.1024、0.8285±0.1945和1.2541±0.5245;积水组与阻塞组相比,积水组与对照组相比有明显差异。
(3)三组患者窗口期子宫内膜组织中GdA基因的表达差异积水组、阻塞组和对照组患者子宫内膜组织中GdA相对表达量依次为
2.0815±0.3425、3.1578±0.6712、和4.1257±0.9476;积水组与对照组相比、阻塞组与对照组相比有明显差异。
4.免疫组织化学实验:
(1)三组患者窗口期子宫内膜组织中OPN的表达差异OPN定位于子宫内膜腺上皮及腔上皮细胞,基质细胞中无表达或仅极少量表达。三组相比无显著性差异。
(2)三组患者窗口期子宫内膜组织中整合素β3的表达差异整合素β3定位于子宫内膜腺上皮细胞和(或)腔上皮细胞,基质细胞仅极少量表达。大部分的输卵管积水患者的子宫内膜上整合素β3表达缺失或呈弱阳性。积水组的表达强度显著低于阻塞组及对照组。
(3)三组患者窗口期子宫内膜组织中GdA的表达差异GdA定位于子宫内膜腺上皮及腔上皮细胞,基质细胞中无表达或仅极少量表达。积水组和阻塞组的表达强度较对照组都明显降低。
结论:
1.输卵管积水患者IVF-ET治疗的临床妊娠率低、早期流产率高;
2.输卵管积水对窗口期子宫内膜多种基因的表达产生影响;其中表达差异最大的基因是酶活性相关基因和信号转导蛋白基因。
3.输卵管积水和输卵管其他病变不影响窗口期子宫内膜OPN基因的表达、转录及其蛋白质的合成分泌;
4.输卵管积水抑制窗口期子宫内膜上整合素β3基因表达、转录及其蛋白合成分泌,降低子宫内膜容受胚胎粘附、着床能力;
5.输卵管积水降低窗口期子宫内膜上GdA基因表达、转录及其蛋白质合成分泌,破坏子宫内膜对胚胎的免疫耐受;
Objective: To study the impaction of hydrosapinx on the outcome of IVF-ET. To explore gene expression changes of hydrosapinx patient in the window of implantation (WOI), demonstrate differentially expressed genes, and explore their clinical significance. Validation of array data was accomplished by mRNA quantification by real time quantitative fluorescent PCR (RT-PCR) and by level of protein expression of some differentially expressed genes. And explore their clinical meaning.
Methods: Statistic Analysis: Two groups composed of patients between August 2003 and August 2005 are built. Hydrosapix group is patients with hydrosapinx treated with IVF-ET for 143 cycles. Tubal obstruction group is patients with other pathologic change without hydrosapinx treated with IVF-ET for 263 cycles. Compare the two groups' IVF-ET outcome.
Oligonucleotide Microarray : The differentially expressed genes (Signal Log Ratio (?)2 or (?)2) of endometrium were analyzed among hydrosapinx, tubal obstruction without hydrospinx and healthy volunteer in WOI, using Affymetrix U133A 2.0 gene chip, Functional classification of differentially expressed genes were established by searching in the human data-base.
RT-PCR : Osteopontin (OPN or SPPI), Placenta protein-14 (PP-14, Glycodelin, GdA or PAEP) and Integrinβ_3, which is differentially expressed or closely related with endometrium receptivity, were verified by PT-PCR. The mRNA quantification of 31 hydrosapinx patients, 29 tubal obstruction patients without hydrosapinx and 21 healthy volunteers were analyzed.
Immunohistochemistry: Analyze and verify the changes of three groups' OPN, GdA and Integrinβ_3 by Immunohistochemistry.
SPSS 13.0 statistic package was used and p<0.05 was adopted as significance level.
Results:
1. The treatment outcome of IVE-ET
The quality of transferred embryo between hydrosapinx and tubal obstruction group has no statistical difference. In the hydrosapinx group, the rates of clinical pregnancy, embryo implantation, early abortion and ectopic pregnancy were 32.87%, 18.61%, 19.15% and 17.02%. In the bubal obstruction group, the rates of clinical pregnancy, embryo implantation, early abortion and ectopic pregnancy were 49.8%, 31.87%, 6.87% and 4.58%.
2. Oligonucleotide Microarray:
(1) The differentially expressed genes
There were 588 genes significantly differentially expressed between hydrosapinx patient and healthy volunteer, 180 genes between tubal obstruction patient and healthy volunteer, 750 genes between hydrosapinx and tubal obstruction patient without hydrosapinx. Those genes were classified into 10 groups according to their main function. They are enzyme, cell adhesion, structural protein, regulation of cell cycle, ion binding protein, signal-transporting proteins, immunomodulators, carrier proteins, unknown function ect.
(2) Differentially expressed genes between hydrosapinx patient and healthy volunteer
Total of 588 genes were significantly differentially expressed. 351 genes were up-regulated, and 237 genes were down-regulated. Enzyme related genes were at most among both up-regulated and down-regulated genes. Signal-transporting proteins were second.
(3) Differentially expressed genes between tubal obstruction patient without hydrosapinx and healthy volunteer
Total of 180 genes were significantly differentially expressed. 106 genes were up-regulated, and 74 genes were down-regulated. Enzyme related genes were at most among significantly differentially expressed genes. Signal-transporting proteins were next.
(4) Differentially expressed genes between hydrosapinx and tubal obstruction patient
Total of 750 genes were significantly differentially expressed. 302 genes were up-regulated, and 448 genes were down-regulated. Signal-transporting proteins were the most among significantly differentially expressed genes except Enzyme.
3.RT-PCR
(1) The different expression of OPN genes mRNA among three groups
The OPN relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 0.6238±0.2690, 0.8955±0.3887 and 1.1065±0.7821. There was no obvious difference between any two groups.
(2) The different expression of Integrinβ_3 genes mRNA among three groups
The Integrinβ_3 relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 0.1945±0.1024, 0.8285±0.1945 and 1.2541±0.5245.There was obvious difference between hydrosapinx group and tubal obstruction group. So was hydrosapinx group and health control group.
(3) The different expression of GdA genes mRNA among three groups
The GdA relative expression quantification among hydrosapinx group, tubal obstruction group and healthy control group were 2.0815±0.3425, 3.1578±0.6712 and 4.1257±0.9476. There was obvious difference between hydrosapinx group and health control group. So was hydrosapinx group and health control group.
4. Immunohistochemistry
(1) The different expression of OPN among three groups
OPN was situated in endometrium glandular epithelium and cavitas epithelial cells. There was little expression in stroma-cell. There was no significant difference among three groups.
(2) The different expression of Integrinβ_3 among three groups
Integrinβ_3 was situated in endometrium glandular epithelium and (or) cavitas epithelial cells. There was only a little expression in stroma-cell. The expression of Integrinβ_3 in most hydrosapinx patients' endometrium is absent or weakly positive. The intension of expression in hydrosapinx group is obviously lower than tubal obstruction group and healthy control group.
(3) The different expression of GdA among three groups
GdA was situated in endometrium glandular epithelium and (or) cavitas epithelial cells. There was little expression in stroma-cell. Both hydrosapinx group and tubal obstruction group are obviously lower than healthy control group.
Conclusion:
1. Hydrosapinx patient treated with IVF-ET has low clinical pregnancy rate, while high early abortion rate.
2. Hydrosapinx has influence on many endometrium genes' expression. The most differentially expressed genes are enzyme related genes and signal-transporting protein genes.
3. Hydrosapinx has no effects on OPN gene expression, transcription, protein synthesis and secretion in WOI endometrium. So does tubal obstruction
4. Hydrosapinx inhibits Integrinβ_3 gene expression, transcription, protein synthesis and secretion in WOI endometrium. So that destroys the ability of endometrium recepting embryo adhesion and implantation.
6. Hydrosapinx inhibits GdA gene expression, transcription, proteinsynthesis and secretion in WOI endometrium. And destroys the immune tolerance of endometrium to embryo.
引文
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