PCOS卵泡颗粒细胞P450芳香化酶、FSHR及LHR表达与卵泡功能的相关性研究
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摘要
目的:1.探讨多囊卵巢综合征(polycystic ovarian syndrome,PCOS)患者与正常妇女卵泡颗粒细胞(granulosa cell,GC)表达P450芳香化酶(cytochrome P450 aromatase,P450arom)的差异及P450arom在卵泡发育中的表现。2.探讨颗粒细胞卵泡刺激素受体(follicle stimulating hormonereceptor,FSHR)、黄体生成素受体(luteinizing hormone receptor,LHR)及P450arom的表达与PCOS卵泡功能的相关性。
方法:本实验选取2007年3月至2007年11月期间在我院生殖中心进行体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)治疗的PCOS患者(PCOS组)及同期非PCOS原因导致不育的患者(正常对照组)共48例。在取卵日分别收集卵泡直径≥14mm(大卵泡组)及＜14mm(小卵泡组)所有卵泡液,同时分离颗粒细胞,Trizol一步法提取颗粒细胞总RNA,进行光密度(OD)定量分析后,P450arom mRNA(messenger RNA,信使RNA),FSHR mRNA及LHR mRNA分别进行逆转录-聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR),PCR产物做琼脂糖凝胶电泳后,用凝胶图像分析仪摄片,进行半定量分析。在取卵日收集血清用免疫化学发光法测定其雌二醇(estradiol,E2)及睾酮(testosterone,T)浓度。收集的≥14mm及＜14mm卵泡液,分别用免疫化学发光法测定其E2浓度并用放射免疫分析技术(radioimmunoassay,RIA)测定其雄烯二酮(androstenedione,A)浓度。
结果:PCOS组与正常对照组相比较,HCG日B超总卵泡数及获卵数均显著增多,Gn(gonadotropin,促性腺激素)用量显著低于正常对照组。PCOS组血清E2及T水平均明显高于正常对照组,差异十分显著(P＜0.01),PCOS组与正常对照组血清E2/T值相比较则无显著性差异(P＞0.05)。卵泡液激素测定结果显示,PCOS研究组中大卵泡组E2水平明显高于小卵泡组(P＜0.05),而在正常组中大卵泡组与小卵泡组比较E2水平差别无统计学意义,分别比较大小卵泡组中PCOS组与正常对照组E2水平均无明显差别;PCOS组与正常对照组的大卵泡组A水平均低于小卵泡组A水平,且在大卵泡组及小卵泡组中,PCOS两组A水平均高于正常对照两组;E2/A值则与之相反,大卵泡组均高于小卵泡组,PCOS组均低于正常对照组,差异有统计学意义。颗粒细胞P450arom mRNA的表达水平在PCOS组和正常对照组中大卵泡组均高于小卵泡组,且PCOS大小卵泡组均低于正常对照两组,差异有统计学意义;颗粒细胞FSHRmRNA的表达水平仅在PCOS组大卵泡组高于小卵泡组,余差别均无统计学意义;颗粒细胞LHRmRNA的表达水平在PCOS组中大卵泡组高于小卵泡组,在正常组中无差别,PCOS组的LHRmRNA的表达水平大小卵泡组均高于正常对照两组,差异有统计学意义。颗粒细胞P450芳香化酶的表达与E2/A的值显著正相关,PCOS组和正常对照组大小卵泡颗粒细胞表达的FSHR与P450芳香化酶也成正相关关系,PCOS组的LHR表达也与P450芳香化酶有正相关性。
结论:1.颗粒细胞P450arom mRNA的表达量随卵泡体积的增大而升高。P450arom的活性随卵泡体积的增大而增强。2.颗粒细胞FSHR及LHR的表达均与P450芳香化酶有一定正相关关系。3.PCOS卵泡颗粒细胞P450arommRNA的表达水平降低,PCOS患者芳香化酶活性不足,较正常女性弱。可能是PCOS发病的一个关键因素,有待进一步证明。
Objective:1.To demonstrate the difference of cytochrome P450 aromatase (P450arom)expression in granulosa cells(GC)between polycystic ovary syndrome(PCOS)patients follicular fluid and normal and the performance of P450arom in follicular Development.2.To explore the relationship between follicle stimulating hormone receptor(FSHR),luteinizing hormone receptor (LHR)and P450arom expression in GC and function of PCOS follicle.
Methods:This study selected from March 2007 to November 2007 in our reproductive centre for in vitro fertilization-Embryo Transfer(IVF-ET)in the treatment patients with PCOS(PCOS group)and the same period non-PCOS causes infertility patients(normal group),a total of 48 cases.Respectively,in the egg retrival collection follicles≥14 mm in diameter(large follicles group)and＜14mm(small follicles group)all follicular fluid,and particle separation granulosa cells,Trizol one-step extraction of total RNA,optical density(OD) quantitative analysis,The amounounts of the mRNA expressions of P450arom and LHR and FSHR in GC respectively were measured by RT-PCR usingβ-actin as intra-control simultaneously.In the oocytes retrival serum collection with immune chemiluminescence method for determination of its estradiol(E2) and testosterone(T)concentration.Collected≥14mm and＜14mm follicular fluid,respectively using immune chemiluminescence method for determination of the E2 and by RIA analysis of its Androstenedione(A).
Result:On the day of injected HCG the total number of follicles and the oocytes retrieval in the women of PCOS group were significantly higher than those in the normal group;The Gn dosage in PCOS group was significantly lower than the normal group.Serum E2 and T level were higher in PCOS group than normal group,the difference is significant(P＜0.01);The value of E2/T in PCOS group and normal group compared with no significant difference(P＞0.05). The levels of E2 in follicular fluid in large follicles group(≥14mm)were significantly higher than that in small follicles group(＜14mm)in PCOS group,whereas in normal group compared E2 levels with no significant difference,respectively compares in large follicles group and small group E2 levels were not significantly different between PCOS group and normal group; The levels of A in follicular fluid in large follicles group(≥14mm)were significantly lower than that in small follicles group(＜14mm)in PCOS group and normal group,and A level in the PCOS group was higher than that in normal group;E2/A value Instead,the large group were higher than those of small follicles group,PCOS group were lower than the control group,there were significant differences.Granulosa tells P450arom mRNA expression levels in large follicles group were higher than those in small follicles group in the PCOS group and the normal group,and the PCOS group were lower than the control group,there were significant differences;The expression level of FSHR mRNA in GC in the large follicles group was higher than that of small follicles group only in PCOS group;The expression level of LHR mRNA in large follicles group were higher than those in small follicles group in PCOS group,no difference in the normal group,the PCOS group LHRmRNA expression level were higher than the normal group in the two large and small groups,there were significant differences.Expression of P450arom mRNA in GC was significantly positively related to E2/A,the expression of LHR and FSHR mRNA also have a positive correlation with expression P450arom mRNA.
Conclusions:1.The expression of P450arom mRNA in GC with the increase of volume increase.P450arom activity with the increase of volume increase.2.The expression of FSHR and LHR and P450arom there is a positive correlation between certain.3.PCOS GC decreased the expression level of P450arommRNA,P450arom activity in PCOS patients lack weaker than normal women.PCOS is a key factor in the pathogenesis of pending further evidence.
方法:本实验选取2007年3月至2007年11月期间在我院生殖中心进行体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)治疗的PCOS患者(PCOS组)及同期非PCOS原因导致不育的患者(正常对照组)共48例。在取卵日分别收集卵泡直径≥14mm(大卵泡组)及＜14mm(小卵泡组)所有卵泡液,同时分离颗粒细胞,Trizol一步法提取颗粒细胞总RNA,进行光密度(OD)定量分析后,P450arom mRNA(messenger RNA,信使RNA),FSHR mRNA及LHR mRNA分别进行逆转录-聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR),PCR产物做琼脂糖凝胶电泳后,用凝胶图像分析仪摄片,进行半定量分析。在取卵日收集血清用免疫化学发光法测定其雌二醇(estradiol,E2)及睾酮(testosterone,T)浓度。收集的≥14mm及＜14mm卵泡液,分别用免疫化学发光法测定其E2浓度并用放射免疫分析技术(radioimmunoassay,RIA)测定其雄烯二酮(androstenedione,A)浓度。
结果:PCOS组与正常对照组相比较,HCG日B超总卵泡数及获卵数均显著增多,Gn(gonadotropin,促性腺激素)用量显著低于正常对照组。PCOS组血清E2及T水平均明显高于正常对照组,差异十分显著(P＜0.01),PCOS组与正常对照组血清E2/T值相比较则无显著性差异(P＞0.05)。卵泡液激素测定结果显示,PCOS研究组中大卵泡组E2水平明显高于小卵泡组(P＜0.05),而在正常组中大卵泡组与小卵泡组比较E2水平差别无统计学意义,分别比较大小卵泡组中PCOS组与正常对照组E2水平均无明显差别;PCOS组与正常对照组的大卵泡组A水平均低于小卵泡组A水平,且在大卵泡组及小卵泡组中,PCOS两组A水平均高于正常对照两组;E2/A值则与之相反,大卵泡组均高于小卵泡组,PCOS组均低于正常对照组,差异有统计学意义。颗粒细胞P450arom mRNA的表达水平在PCOS组和正常对照组中大卵泡组均高于小卵泡组,且PCOS大小卵泡组均低于正常对照两组,差异有统计学意义;颗粒细胞FSHRmRNA的表达水平仅在PCOS组大卵泡组高于小卵泡组,余差别均无统计学意义;颗粒细胞LHRmRNA的表达水平在PCOS组中大卵泡组高于小卵泡组,在正常组中无差别,PCOS组的LHRmRNA的表达水平大小卵泡组均高于正常对照两组,差异有统计学意义。颗粒细胞P450芳香化酶的表达与E2/A的值显著正相关,PCOS组和正常对照组大小卵泡颗粒细胞表达的FSHR与P450芳香化酶也成正相关关系,PCOS组的LHR表达也与P450芳香化酶有正相关性。
结论:1.颗粒细胞P450arom mRNA的表达量随卵泡体积的增大而升高。P450arom的活性随卵泡体积的增大而增强。2.颗粒细胞FSHR及LHR的表达均与P450芳香化酶有一定正相关关系。3.PCOS卵泡颗粒细胞P450arommRNA的表达水平降低,PCOS患者芳香化酶活性不足,较正常女性弱。可能是PCOS发病的一个关键因素,有待进一步证明。
Objective:1.To demonstrate the difference of cytochrome P450 aromatase (P450arom)expression in granulosa cells(GC)between polycystic ovary syndrome(PCOS)patients follicular fluid and normal and the performance of P450arom in follicular Development.2.To explore the relationship between follicle stimulating hormone receptor(FSHR),luteinizing hormone receptor (LHR)and P450arom expression in GC and function of PCOS follicle.
Methods:This study selected from March 2007 to November 2007 in our reproductive centre for in vitro fertilization-Embryo Transfer(IVF-ET)in the treatment patients with PCOS(PCOS group)and the same period non-PCOS causes infertility patients(normal group),a total of 48 cases.Respectively,in the egg retrival collection follicles≥14 mm in diameter(large follicles group)and＜14mm(small follicles group)all follicular fluid,and particle separation granulosa cells,Trizol one-step extraction of total RNA,optical density(OD) quantitative analysis,The amounounts of the mRNA expressions of P450arom and LHR and FSHR in GC respectively were measured by RT-PCR usingβ-actin as intra-control simultaneously.In the oocytes retrival serum collection with immune chemiluminescence method for determination of its estradiol(E2) and testosterone(T)concentration.Collected≥14mm and＜14mm follicular fluid,respectively using immune chemiluminescence method for determination of the E2 and by RIA analysis of its Androstenedione(A).
Result:On the day of injected HCG the total number of follicles and the oocytes retrieval in the women of PCOS group were significantly higher than those in the normal group;The Gn dosage in PCOS group was significantly lower than the normal group.Serum E2 and T level were higher in PCOS group than normal group,the difference is significant(P＜0.01);The value of E2/T in PCOS group and normal group compared with no significant difference(P＞0.05). The levels of E2 in follicular fluid in large follicles group(≥14mm)were significantly higher than that in small follicles group(＜14mm)in PCOS group,whereas in normal group compared E2 levels with no significant difference,respectively compares in large follicles group and small group E2 levels were not significantly different between PCOS group and normal group; The levels of A in follicular fluid in large follicles group(≥14mm)were significantly lower than that in small follicles group(＜14mm)in PCOS group and normal group,and A level in the PCOS group was higher than that in normal group;E2/A value Instead,the large group were higher than those of small follicles group,PCOS group were lower than the control group,there were significant differences.Granulosa tells P450arom mRNA expression levels in large follicles group were higher than those in small follicles group in the PCOS group and the normal group,and the PCOS group were lower than the control group,there were significant differences;The expression level of FSHR mRNA in GC in the large follicles group was higher than that of small follicles group only in PCOS group;The expression level of LHR mRNA in large follicles group were higher than those in small follicles group in PCOS group,no difference in the normal group,the PCOS group LHRmRNA expression level were higher than the normal group in the two large and small groups,there were significant differences.Expression of P450arom mRNA in GC was significantly positively related to E2/A,the expression of LHR and FSHR mRNA also have a positive correlation with expression P450arom mRNA.
Conclusions:1.The expression of P450arom mRNA in GC with the increase of volume increase.P450arom activity with the increase of volume increase.2.The expression of FSHR and LHR and P450arom there is a positive correlation between certain.3.PCOS GC decreased the expression level of P450arommRNA,P450arom activity in PCOS patients lack weaker than normal women.PCOS is a key factor in the pathogenesis of pending further evidence.
引文
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11 Araki H,Tsubota T,Maeda N,et al.Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season;Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season.J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science;J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science,1996,58;58(8;8):787-90;787-90.
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22 Willis D,Mason H,Gilling-Smith C,et al.Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries.J Clin Endocrinol Metab JT-The Journal of clinical endocrinology and metabolism,1996,81(1):302-9.
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24 袁鹰,鲁梅芳,王箐。肥胖并子宫内膜癌病人腹部脂肪组织细胞色素P450芳香化酶mRNA表达.青岛大学医学院学报,2004,4(3):208-210.
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20 Araki H,Tsubota T,Maeda N,et al.Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season;Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season.J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science;J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science,1996,58;58:787-90;787-90.
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7 Mahendroo MS,Means GD,Mendelson CR,et al.Tissue-specific expression of human P-450AROM.The promoter responsible for expression in adipose tissue is different from that utilized in placenta.J Biol Chem JT-The Journal of biological chemistry,1991,266(17):11276-81.
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10 Hunter MH,Sterrett JJ.Polycystic ovary syndrome:it's not just infertility.Am Faro Physician JT-American family physician,2000,62(5):1079-88,1090.
11 Araki H,Tsubota T,Maeda N,et al.Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season;Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear,Ursus arctos yesoensis during the mating season.J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science;J Vet Med Sci JT-The Journal of veterinary medical science/the Japanese Society of Veterinary Science,1996,58;58(8;8):787-90;787-90.
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13 Stevenson AF.Human granulosa cells in vitro:characteristics of growth,morphology and influence of some cytokines on steroidogenesiso Indian J Exp Biol JT-Indian journal of experimental biology,2000,38(12):1183-91.
14 Agarwal SK,Judd HL.Estrogen replacement therapy and breast cancer.Fertil Steril JT-Fertility and sterility,1999,71(4):602-3.
15 Vrbikova J,Hill M,Starka L,et al.The effects of long-term metformin treatment on adrenal and ovarian steroidogenesis in women with polycystic ovary syndrome.Eur J Endocrinol JT-European journal of endocrinology/European Federation of Endocrine Societies,2001,144(6):619-28.
16 la Marca A,Morgante G.Palumbo M,et al.Insulin-lowering treatment reduces aromatase activity in response to follicle-stimulating hormone in women with polycystic ovary syndrome.Fertil Steril JT-Fertility and sterility,2002,78(6):1234-9.
17 Jakimiuk AJ,Weitsman SR,Brzechffa PR,et al.Aromatase mRNA expression in individual follicles from polycystic ovaries.Mol Hum Reprod JT-Molecular human reproduction,1998,4(1):1-8.
18 Deshpande RR,Chang MY,Chapman JC,et al.Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection.Am J Reprod Immunol JT-American journal of reproductive immunology(New York,N.Y:1989),2000,44(2):80-8.
19 Wilson EA,Erickson GF,Zarutski P,et al.Endocrine studies of normal and polycystic ovarian tissues in vitro.Am J Obstet Gynecol JT-American journal of obstetrics and gynecology,1979,134(1):56-63.
20 Erickson GF,Magoffin DA,Cragun JR,et al.The effects of insulin and insulin-like growth factors-Ⅰ and -Ⅱ on estradiol production by granulosa cells of polycystic ovaries.J Clin Endocrinol Metab JT-The Journal of clinical endocrinology and metabolism,1990,70(4):894-902.
21 Coffler MS,Patel K,Dahan MH,et al.Evidence for abnormal granulosa cell responsiveness to follicle-stimulating hormone in women with polycystic ovary syndrome.J Clin Endocrinol Metab JT-The Journal of clinical endocrinology and metabolism,2003,88(4):1742-7.
22 Willis D,Mason H,Gilling-Smith C,et al.Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries.J Clin Endocrinol Metab JT-The Journal of clinical endocrinology and metabolism,1996,81(1):302-9.
23 Greisen S,Ledet T,Ovesen P.Effects of androstenedione,insulin and luteinizing hormone on steroidogenesis in human granulosa luteal cells.Hum Reprod JT-Human reproduction(Oxford,England),2001,16(10):2061-5.
24 袁鹰,鲁梅芳,王箐。肥胖并子宫内膜癌病人腹部脂肪组织细胞色素P450芳香化酶mRNA表达.青岛大学医学院学报,2004,4(3):208-210.