【作者】 曾晓霞；

【导师】 刘风华；

【作者基本信息】 广州医学院 ， 妇产科学， 2011， 硕士

【摘要】 研究背景多囊卵巢综合征（polycystic ovary syndrome,PCOS）是导致育龄妇女无排卵性不孕的常见原因,发病率约4%¹2%。其临床表现呈高度异质性,常表现为月经不规律,肥胖、多毛,伴胰岛素抵抗及高胰岛素血症。PCOS的特点由其发生机制所决定,该机制导致下丘脑—垂体—卵巢—肾上腺各部位功能的异常,并伴有代谢异常。相关的代谢功能失调包括:胰岛素抵抗、血脂异常以及日益增加的肥胖问题。正常排卵周期是由性腺以及卵巢内、外的各种生长因子所调节,这些因子的异常被推测在PCOS病理生理中起重要作用。生长激素（growth hormone,GH）及其相关因子对下丘脑—垂体—卵巢轴具有重要的调节作用,影响卵巢的成熟及卵泡的周期性发育。近年国外有研究认为PCOS患者GH-IGF轴功能混乱,学者们对PCOS患者相对正常对照组基础GH分泌有降低、正常、升高,观点不一,但大部分研究认为PCOS患者对左旋多巴及GHRH刺激的反应迟钝,分泌受损,可能与肥胖、胰岛素抵抗、高雄激素血症有关,确切机制尚不清楚。生长激素具有促进细胞增殖、蛋白质合成、调理免疫功能等作用,已在辅助生殖领域应用已久,常用于卵巢反应不良患者辅助治疗,有助于改善助孕结局。因此,探讨PCOS及卵巢反应不良患者GH及IGF-1的水平与妊娠结局的关系及其对预测卵巢反应的价值具有重要的临床指导意义。目的检测GH、IGF-1在PCOS及卵巢反应不良患者表达水平,并结合临床指标评价GH、IGF-1对PCOS及卵巢反应不良患者行体外受精-胚胎移植（in vitro fertilization-embryo transfer, IVF-ET）结局的影响,探讨GH、IGF-1对预测卵巢反应的价值。方法对2010年3月至2010年10月于广州医学院第三附属医院生殖中心行体外受精-胚胎移植的65名患者进行前瞻性病例对照研究。将PCOS患者作为研究组,共29例,同期的输卵管因素不孕患者作为对照组,共36例,另根据获卵数分成低反应（≤4）及正常反应（>4）两个亚组。没有一例获卵数超过30个。所有患者均采用黄体期长方案降调节促排卵,于取卵日收集两组患者外周静脉血及卵泡液,采用时间分辨免疫荧光法（time-resolved fluoroimmunoassay,TRFIA）和酶联免疫吸附法（enzyme linked immunosorbent assay,ELISA）分别测定两组患者血清及卵泡液中GH,IGF-1的浓度。对三组间的IVF-ET结局:获卵数、受精率、优质胚胎率、种植率、临床妊娠率进行比较,并结合GH、IGF-1水平进行相关性分析。结果1、所有患者血清GH及IGF-1水平较卵泡液高（ GH 9.01+1.33 VS.2.97±1.75;P=0.000, IGF-1 111.89±28.54VS.90.19±19.65;P=0.000）,PCOS患者血清GH显著高于低反应组（P<0.001）,与正常反应组无差异,卵泡液GH在各组无显著性差异;血清及卵泡液IGF-1水平三组间两两比较均有显著性差异,PCOS患者高于正常反应组,正常反应组高于低反应组（P<0.001）;2、三组间子宫内膜厚度无统计学差异（P>0.05）;HCG日E2、Gn用量、获卵数、2PN胚胎数及卵裂数在三组间两两比较均有显著性差异（P<0.05）,PCOS组高于正常反应组,正常反应组高于低反应组;PCOS组优质胚胎数高于正常反应组及低反应组,均有统计学差异（P<0.05）;三组间种植率有显著性差异（P<0.05）,正常反应组最高,低反应组最低;受精率、妊娠率、临床妊娠率、继续妊娠率、流产率、OHSS发生率及周期取消率均无统计学差异（P>0.05）。3、血清和卵泡液中IGF-1水平与Gn用量呈负相关趋势,与获卵数、受精数、卵裂数、优质胚胎数、2PN数、种植率呈正相关趋势,卵泡液GH与种植率呈正相关趋势,均有统计学差异（P<0.05）;血清和卵泡液中GH水平与获卵数、受精数、卵裂数、优质胚胎数、2PN数呈正相关趋势,但均无统计学差异（P>0.05）。4、血清和卵泡液GH及血清IGF-1水平在妊娠组和非妊娠组比较无统计学差异（P>0.05）;卵泡液IGF-1在妊娠组明显高于非妊娠组,差异有统计学意义（P<0.05）。结论血清GH、IGF-1水平较卵泡液高,PCOS患者取卵日GH、IGF-1与对照组比较并无降低,但低反应组血清及卵泡液GH、IGF-1水平均较低,血清和卵泡液中IGF-1水平与Gn用量呈负相关趋势,与获卵数、受精数、卵裂数、优质胚胎数、2PN数、种植率呈正相关趋势,卵泡液GH与种植率呈正相关趋势,血清和卵泡液中GH水平与获卵数、受精数、卵裂数、优质胚胎数、2PN数无相关性,妊娠组卵泡液IGF-1水平高于非妊娠组。表明PCOS患者在促排卵过程中并无GH、IGF-1分泌减少,卵泡液中的GH、IGF-1主要由循环分泌渗透而来,由于GH的易受其他因素影响,其作为预测卵巢反应的价值有待进一步研究,IGF-1稳定性较好,可以作为预测卵巢反应的指标之一。目的了解人卵泡黄素化颗粒细胞中生长激素（GH）和胰岛素样生长因子-1（IGF-1）受体的表达,GH在体外对人卵泡黄素化颗粒细胞产生雌、孕激素的影响。方法采用免疫细胞化学染色法,测定24例行体外受精-胚胎移植治疗患者的卵泡黄素化颗粒体细胞的GH和IGF-1受体表达,并将黄素化颗粒细胞在不同浓度的GH（0、0.1、0.2、0.4mg/m1）作用下单独及与促卵泡素（FSH,浓度为75U/ml）共同培养72h,采用酶联免疫吸附法测定黄素化颗粒细胞培养液中的IGF-1和化学发光法测定雌二醇和孕酮含量。结果80%以上黄素化颗粒细胞GH和IGF-1受体阳性;黄素化颗粒细胞在基础状态下能自分泌一定量的IGF-1（140.00±27.13ng/ml）,E₂ 444.67±67.25（pg/ml）,和P（655.00±70.07ng/ml）,GH不能直接刺激黄素化颗粒细胞合成IGF-1,E₂和P,但能直接或间接通过IGF-1协同FSH进一步促进E₂和P的合成,且呈剂量依赖性。更多还原

【Abstract】 BackgroundPCOS is one of the most common endocrine disorders in women of reproductive age, with prevalence ranging from 4% to 12%.Polycystic ovary syndrome is a heterogeneous clinical condition, characterized by hirsutism, irregular menstrual cycles, infertility, and endocrine abnormalities such as hyperandrogenism. Moreover, a considerable percentage of women with PCOS present insulin resistance and compensatory hyperinsulinemia. Women with PCOS have a disturbance of normal GH kinetics. Serum GH baseline concentrations in women with PCOS have been variously described as low,normal or elevated, while the diurnal GH secretion was found to be impaired. It has been shown that women with PCOS have a relative deficiency or reduction in GH reserve after oral L-dopa stimulation. The GH concentrations after GHRH administration were found to be impaired in women with PCOS. A number of recent clinical studies have also suggested alterations in the GH/IGF-I system in women with PCOS.Therefore,The purpose of the present study was to evaluate the serum and intrafollicular concentrations of GH and IGF-1 and its relationship with the outcomes of women with polycystic ovary syndrome and low ovrian responders undergoing IVF-ET. To determine if these factors are predictive of IVF outcome. ObjectiveTo evaluate the serum and intrafollicular concentrations of GH and IGF-1 and its relationship with the outcomes of women with polycystic ovary syndrome and low ovrian responders undergoing IVF-ET. Investigate if these factors are predictive of IVF outcome.MethodsA prospective case-control study was conducted on 65 patients who were submitted to ovulation induction for IVF-ET. These patients were divided into the study group（29 patients with endometriosis） and the control group（36 patients with tubal factor infertility）.The control patients were subgroup as low and norm ovarian response according to the number of oocytes retrieved. Whose numbers of oocytes retrieval was less than 4 was defined as low responders.No one in norm ovarian response group had more than 30 oocytes retrieved. All patients underwent a long GnRH agonist protocol for pituitary downregulation followed by controlled ovarian hyperstimulation. Peripheral blood and follicular-fluid samples were collected on the day of oocyte retrieval. Growth hormone（GH）, Insulin-like growth factor-1（IGF-1） were measured by time-resolved fluoroimmunoassay （TRFIA） and enzyme linked immunosorbent assay （ELISA） respectively. Compare the outcome of IVF-ET between the three groups: include retrieved oocytes, fertilization rates, good-embryo rates, implantation rates and clinical pregnancy rates, and analysis the correlation between the levels of above growth factors and the outcome of IVF-ET.Results1.The concentrations of GH and IGF-1 in serum was significantly higher than follicular fruid（9.01+1.33 VS.2.97±1.75;P=0.000, 111.89±28.54VS.90.19±19.65;P=0.000,respectively）.Serum GH level in PCOS patients was higher than low ovarian responsers（P=0.000）,but norm ovarian responsers had no significantly differences comparing with the other two groups. Follicular fruid GH levels were no significantly differences among three groups.The serum and follicular fruid IGF-1 levels had significantly differences between each other in the three groups（P<0.001）.2.Endometrial thickness had no significantly differences between each other（P>0.05）. Peak E2 levels on the day of HCG injection, doses of gonadotropin requirement,the number of oocytes retrieved,cleavage embroys and 2PN were significantly differences between each other（P<0.05）. PCOS womem had more high-quailty embryos than normal and low ovarian responsers. Implantation rates was differences among three groups（P=0.007）,implantation rate in low ovarian responsers was lowest ,while the fertilization rates,the pregnancy rates,clinical pregnancy rates,ongoing pregnancy rates,cancellation rates and OHSS rates were lower and spontaneous abortion rate was higher in poor ovarian responsers but had no significantly differences among three groups.3. No relationship was found between the concentration of GH in serum or follicular fruid and the number of oocytes retrieved,high-quailty embryos,fertilizaton,cleavage embroys , as well as 2PN embroys （P>0.05）, However, follicular fruid GH was correlated with implantation rates,while serum and follicular fruid IGF-1 levels were correlated with oocytes retrieved,high-quailty embryos,fertilizaton rate,cleavage embroys,2PN embroys and implantation rates （P>0.05）.4. GH in serum or follicular fruid as well as IGF-1 in serum had no significantly differences between pregnancy group and non pregnancy group,but IGF-1 in follicular fruid had significantly differences between the two groups.ConclusionsSerum GH and IGF-1 concentrations were statistically significantly higher than in follicular fruid. It is thought that the origin of follicular fruid GH and IGF-1 is through transudation of serum GH and IGF-1 derived from an extraovarian source and through local production in the granulosa cells.Our result fail to show there was difference in the concentrations of GH in serum and follicular fruid between PCOS and normal response patient on day of oocytes retrieved,but these factors were lowest in poor ovarian responsers. No relationship was found between the concentration of GH in serum or follicular fruid and the number of oocytes retrieved,high-quailty embryos,fertilizaton,cleavage embroys , as well as 2PN embroys . However, follicular fruid GH was correlated with implantation rates,while serum and follicular fruid IGF-1 levels were correlated with oocytes retrieved,high-quailty embryos,fertilizaton rate,cleavage embroys,2PN embroys and implantation rates.It means the patients with PCOS didn’t have the diurnal GH、IGF-1 secretion impaired on day of oocyte retrieval,IGF-1 is a good predictor of the ovarian response to COH and IVF outcome.Due to GH secrete characteristic, we need further research to investigate its value as predictor of the ovarian response to COH and IVF outcome. Objective The aim of this study was to determine whether there were differences in the dose-dependent effects of growth hormone（GH） on estradiol（E₂）,progesterone（P）,insulin-like growth factor-1（IGF-1） production by luteinized granulosa cells and to detect GH and IGF-1 receptors in human luteinized granulosa cells. Methods Human luteinized granulosa cells（LGC） were isolated from 24 women undergoing in vitro fertilization and embryo transfer （IVF-ET） .Some of the LGCs were used for detecting GH and IGF-1 receptors by immunocytochemistry（SABC method）.Most of them were cultured in DMEM/F12 medium with 10% fetal bovine serum（FCS）, after 2 days the medium was removed and the cells were incubated with various concentration of GH （0,0.1,0.2 and 0.4mg/ml） in the presence or absence of follicle stimulating hormone（ FSH 75IU/ml）.Media were collected after 72 hours and E₂,P in media were measured with chemiluminescence immune assay（CLIA）while IGF-1 was measured with enzyme linked immunosorbent assay（ELISA）. Results More than 80% of the LGCs were positively stained for GH and IGF-1 receptors . LGCs can autocrine IGF-1,E₂,P at the level of 140.00±27.13ng/ml, 444.67±67.25pg/ml, 655.00±70.07ng/ml without GH or FSH co-culture,GH alone can’t directly stimulate LGCs biosynthesis IGF-1,E₂,and P. However, the combination of GH and FSH can enhance gonadotropin-stimulated steroidogenesis in a significant dose-dependent manner. Conclusions: The main function of GH appeared to synergize with FSH and amplify gonadotropin action and thereby facilitation of granulosa cell replication, differentiation and steroidogenesis directly or indirectly via IGF-1.更多还原