宫内节育器对大鼠子宫内膜血管内皮生长因子及其受体表达影响的研究

目的:研究置固定式铜宫内节育器(FCu-IUD)和固定式吲哚美辛铜宫内节育器(FICu-IUD)的大鼠子宫内膜组织中血管内皮生长因子(VEGF)及其受体Flk-1的表达。方法:采用蛋白印迹法(Western blotting)检测FCu-IUD组、FICu-IUD组大鼠子宫内膜组织VEGF及Flk-1的表达,逆转录聚合酶联反应(RT-PCR)检测各组VEGF mRNA的表达。以未置器一侧作为对照。结果:大鼠置器侧与对照侧子宫内膜均可检测到与VEGF121和VEGF165两种VEGF亚型相对应的mRNA与蛋白表达。FCu-IUD组大鼠置器侧子宫内膜VEGF121及VEGF165表达在mRNA水平和蛋白水平均显著升高,与对照侧比较差异有统计学意义(P<0.001,P<0.05)。置器侧Flk-1蛋白的表达明显高于对照侧(P<0·01)。FICu-IUD组置器侧与对照侧比较,VEGF、Flk-1蛋白及VEGF mRNA表达差异均无显著性。结论:Cu-IUD引起的子宫异常出血可能与VEGF的增加有关,吲哚美辛减少Cu-IUD引起的子宫异常出血的作用可能与通过抑制前列腺素合成从而使VEGF生成减少有关。     

HOXA10 protein expression in the endometrium of normally menstruating women after receiving GnRH antagonist

Objective(s)

To compare HOXA10 protein expression in the endometrium between natural control cycles and GnRH antagonist-treated cycles obtained during the window of implantation of normally menstruating women.

Study design

This study was conducted at the Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Thirty-five volunteers were recruited into this prospective, self-controlled study, which was divided into two cycles, the first a natural control cycle and the second a GnRH antagonist-treated cycle. The two cycles were separated by one resting cycle. In the GnRH antagonist-treated cycle, when the leading follicle was 15 mm, ganirelix (Orgalutran^®) 0.25 mg was administered daily. In both cycles, ovulation was induced when the largest follicle reached 18 mm in diameter. Finally, endometrial biopsy was performed on day 6 after documented ovulation, which corresponds to the window of implantation. Endometrial HOXA10 protein expression, a marker of endometrial receptivity, was analyzed by immunohistochemistry. The protein expression was compared between the two cycles regarding their percentage of immunostained cells and IHC-scores (percentage of stained cells × intensity of nuclear staining).

Results

HOXA10 protein was exclusively localized in the stromal compartment of the endometrium. The percentage of HOXA10 nuclear staining in the endometrium collected from GnRH antagonist-treated cycles was higher than that of the natural cycles, whereas the IHC-scores showed no difference between the two cycles.

Conclusion(s)

GnRH antagonists may have no effect on HOXA10 protein expression in the endometrium obtained during the implantation window of normally menstruating women.     

The effects of GnRH antagonist on the endometrium of normally menstruating women

Purpose

To study the effects of GnRH antagonist (ganirelix-Orgalutran®) on the endometrium of regularly menstruating women.

Materials and methods

Prospective, self-controlled study. The thirty-five volunteers were studied for two cycles: one as a control and the other, GnRH antagonist-treated cycles in which ganirelix 0.25 mg/d was given daily for 3 days, starting when the largest follicle reached 15 mm. In both cycles, serum estradiol, LH and endometrial thickness were measured when the largest follicle was ≥18 mm. Endometrial biopsy was performed on day 6 after ovulation for histological dating and morphometric study.

Results

No statistical differences between histological dating and the endometrial thickness in the control and GnRH antagonist-treated cycles. All morphometric parameters were also not different. Serum estradiol and LH levels were significantly lower in GnRH antagonist-treated cycles.

Conclusion

GnRH antagonist has no effect on the endometrium of regularly menstruating women as assessed by either histological dating or morphometric analysis.

     

Immunohistochemical detection of transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor expression in hyperstimulated rat ovary

OBJECTIVE: The aim of the present study is to figure out the immunohistochemical expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) in hyperstimulated rat ovaries. METHODS: Twenty Wistar-Albino adult female rats (250-300 g) were taken into the study. The animals were randomly divided into two groups, each containing 10 rats: (i) stimulation group and (ii) control group. In the stimulation group, a stimulation regimen was administered to induce follicular maturity and ovarian hyperstimulation syndrome (OHSS) at the end using a 30-IU follicle-stimulating hormone that was administered subcutaneously for 4 consecutive days, followed by a 30-IU human chorionic gonadotropin on day 5 to induce ovulation. The rats, in the control group, received 0.2 ml of 0.9% NaCl for 5 consecutive days to mimic the conditions of the study animals. At the end of the treatment period, all rats underwent ovariectomy and the sections of ovaries were stained for the TGF-alpha, EGF, and VEGF. RESULTS: The expression of TGF-alpha, EGF, and VEGF in the endothelium, the stroma, the granulosa cells, and the corpus luteum was found to be significantly higher in the stimulated group, compared to that in the control group ( p < 0.05). CONCLUSION: TGF-alpha, EGF, and VEGF are found to have increased in the hyperstimulated ovaries and this finding seems to be involved in the OHSS pathogenesis.     

Epidermal growth factor receptor expression in normal and malignant endometrium

Epidermal growth factor receptor expression in fresh-frozen uterine tissues was studied with the use of monoclonal antibody 528, which recognizes an epitope on the external domain of the epidermal growth factor receptor. Immunohistochemically detectable epidermal growth factor receptor was seen in all uterine cell types in 19 of 20 normal uteri. Staining of endometrial glands and endometrial stromal cells was consistently greater than that of myometrium, and no variation in intensity or distribution of staining was seen during the menstrual cycle. Immunohistochemically detectable epidermal growth factor receptor was found less frequently in endometrial adenocarcinomas than in normal endometrium (p less than 0.01). Thirteen of 40 endometrial adenocarcinomas (32.5%) did not express detectable receptor. Epidermal growth factor receptor expression did not correlate with histologic grade, depth of myometrial invasion, estrogen-progesterone receptor status, the presence of extrauterine metastases, or the development of recurrent disease.     

In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix

The distribution of receptors for insulin and epidermal growth factor along the longitudinal axis of the uterine cavity was studied in 28 uteri obtained from women of reproductive age undergoing hysterectomy for benign conditions. Insulin binding to crude plasma membranes was higher (p less than 0.05) in the secretory than in the proliferative phase of the menstrual cycle in all uterine segments (fundus to cervix). Epidermal growth factor binding did not change during the menstrual cycle but the number of epidermal growth factor binding sites was higher in the cervix than in the fundus (p less than 0.05). Scatchard plots of binding data, obtained with crude plasma membranes from pooled uteri, were curvilinear; the high-affinity sites had dissociation constants of 1 to 4 nmol/L and receptor concentrations of 100 to 300 fmol/mg of protein, for both iodine 125-labeled insulin and 125I-labeled epidermal growth factor. In plasma membranes, obtained from another 15 uteri, mouse nerve growth factor (3.3 micrograms/ml) decreased the binding of insulin by an average of 17% (p less than 0.005); in the decidua of a pregnant uterus at 12 weeks Scatchard analysis showed that nerve growth factor decreased the affinity but not the number of insulin-binding sites. Nerve growth factor had no effect on epidermal growth factor binding. Human prolactin (2 micrograms/ml) also decreased insulin binding by an average of 18% (n = 5, p less than 0.025) but had no effect on epidermal growth factor binding. These "baseline" data will be useful in further studies of the possible interactions between (1) receptors for various peptide growth factors and (2) sex steroid hormones, in normal and neoplastic endometrium and cervix.     

The expression of insulin-like growth factor and insulin-like growth factor binding protein mRNAs in mouse placenta

Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are paracrine regulators of tissue growth and development, and are expressed at the sites of biological action. To study the role of the IGFs and IGFBPs in mouse placental development, we determined the temporal and spatial expression patterns of the mRNAs at embryonic days 10.5 to 18.5 by in situ hybridization. IGF-II mRNA was expressed strongly in mesoderm and fetal blood vessels of early placenta and in labyrinthine trophoblast of later placenta. In the junctional zone, IGF-II mRNA was expressed first in spongiotrophoblasts, later strongly in glycogen cells and variably in giant cells. IGFBP-2 mRNA was expressed weakly in spongiotrophoblasts and glycogen cells. IGFBP-2, -5 and -6 mRNAs were detected in the stroma of the metrial gland. Myometrium expressed IGFBP-2 mRNA strongly, IGFBP-6 mRNA moderately and IGFBP-5 mRNA weakly. The endothelium of maternal blood vessels in decidua expressed IGFBP-3 and -5 mRNAs, and some deeper vessels expressed IGFBP-4 mRNA. In the yolk sac, IGF-II mRNA was expressed in endoderm and mesoderm, whereas IGFBP-1, -2 and -4 mRNAs were expressed only in endoderm, and IGFBP-4 mRNA in mesoderm. Strong expression of IGF-II mRNA in glycogen cells suggests a role in the autocrine/paracrine regulation of invasion. Similar to rat and guinea pig, but in contrast to man and primates, IGFBP mRNAs, except IGFBP-4, were not expressed in mouse decidua. However, IGFBP-3, -4 and -5 mRNAs were expressed in endothelium of maternal blood vessels, and IGFBP-2 and -6 mRNAs in myometrium, where IGFBPs may play a critical role in regulating trophoblast invasion. These findings suggest possible biological roles of the peptides at the feto-maternal interface.     

原因不明不孕患者子宫内膜胰岛素样生长因子Ⅱ及其受体mRNA的表达及其与性激素水平的相关性

目的探讨胰岛素样生长因子Ⅱ(IGF-Ⅱ)及其Ⅰ型受体(IGF-ⅠR)、Ⅱ型受体(IGF-ⅡR) mRNA在原因不明不孕患者黄体中期子宫内膜的表达,及其与性激素水平变化的相关性.方法采用原位杂交、逆转录PCR(RT-PCR)技术,检测38例原因不明不孕患者(研究组)和20例正常育龄女性志愿者或因男方因素不孕患者(对照组)黄体中期子宫内膜IGF-Ⅱ、IGF-ⅠR、IGF-ⅡR mRNA的表达;放射免疫法测定同期血清雌二醇、孕酮水平.结果黄体中期子宫内膜间质细胞胞浆内,可见IGF-Ⅱ、IGF-ⅡR蓝色颗粒弥散分布,IGF-ⅠR 则主要在上皮细胞中分布.研究组IGF-Ⅱ mRNA的表达(灰度值,下同)为0.71±0.34,对照组为0.96±0.34,两组比较,差异有显著性(P<0.05);研究组IGF-ⅠR mRNA的表达为0.62±0.28,显著低于对照组的0.93±0.51(P<0.05);研究组IGF-ⅡR mRNA的表达为0.49±0.27,也显著低于对照组的0.73±0.36(P<0.05).研究组黄体中期孕酮水平为(34±15)nmol/L,显著低于对照组(53±17)nmol/L(P<0.01), 而血清雌二醇水平与对照组比较,差异无显著性(P>0.05).研究组与对照组IGF-Ⅱ mRNA与IGF-ⅠR、IGF-ⅡR mRNA的表达呈正相关;两组妇女孕酮与IGF-Ⅱ、IGF-ⅠR mRNA表达水平呈正相关;对照组孕酮与IGF-ⅡR mRNA表达水平呈正相关.结论原因不明不孕患者黄体中期孕酮不足,可能影响子宫内膜IGF-Ⅱ、IGF-ⅠR、IGF-ⅡR mRNA的表达,并可能是导致不孕的病因之一.     

Epidermal growth factor receptor expression in normal endometrium and endometriosis: an immunohistochemical study.

OBJECTIVE: To study epidermal growth factor (EGF) receptor expression in endometrium throughout the menstrual cycle and to compare EGF-receptor expression in endometrium from patients with endometriosis with receptor expression in synchronously sampled endometriosis and in endometrium from healthy women. DESIGN: An immunohistochemical study of receptor expression using murine monoclonal antibodies and timed endometrial and endometriotic biopsies. SUBJECTS: 25 healthy women and 27 patients with a diagnosis of endometriosis. RESULTS: Positive staining for EGF receptors was observed in 24 of 25 samples from normal women and in 26 of 27 endometrial samples from patients with endometriosis. In neither group was there any variation in the intensity of staining throughout the menstrual cycle and both glands and stroma were stained. EGF-receptor expression was observed in the glands of 15 out of 17 endometriotic lesions and in 12 of these biopsies positive staining was also present within endometriotic stroma. CONCLUSION: This study shows no difference in the intensity of staining of EGF receptors in endometrium throughout the menstrual cycle or between the glands of normal endometrium and those of endometriosis.     

Effect of corticotropin-releasing factor receptor antagonist on psychologically suppressed masculine sexual behavior in rats

IntroductionCorticotropin‐releasing factor (CRF) coordinates various responses of the body to stress, and CRF receptors are important targets of treatment for stress‐related disorders.AimTo investigate the effect of a nonselective CRF receptor antagonist, astressin, on suppression of masculine sexual behavior by psychological stress in rats.MethodsFirst, we investigated the influence of psychological stress, induced 2 hours per day for three consecutive days, on sexual behavior. Then, rats were divided into 4 groups: a control group, an astressin administration group (A), a psychological stress loading group (PS), and a psychological stress loading and astressin administration group (PS + A). The rats were exposed to sham or psychological stress for three consecutive days. After the last stress loading, the rats were injected with vehicle or astressin, and their sexual behavior was observed. We also measured serum levels of adrenocorticotropic hormone (ACTH).Main Outcome MeasureThe effects of astressin on sexual behavior and serum levels of ACTH in rats affected by psychological stress were determined.ResultsSexual behavior was reduced after psychological stress loading. The PS rats had significantly longer mount, intromission, and ejaculation latencies and lower ejaculation frequency than did the control, A, and PS + A rats. The intromission latency and ejaculation frequency in the PS + A rats did not achieve the level observed in the controls. There was no significant difference in these parameters between the control and A rats. Serum ACTH levels were significantly lower in PS + A rats than in PS rats.ConclusionsPsychologically suppressed masculine sexual behavior could be partially recovered with astressin administration in rats. These data provide a rationale for the further study of CRF receptor antagonists as novel agents for treating psychological sexual disorders. Miwa Y, Nagase K, Oyama N, Akino H, and Yokoyama O. Effect of corticotropin‐releasing factor receptor antagonist on psychologically suppressed masculine sexual behavior in rats.     

Effect of in vivo GnRH agonist and GnRH antagonist on hCG and insulin-stimulated progesterone production by human granulosa-lutein cells in vitro

Purpose : To investigate hCG and insulin-stimulated progesterone (P) production by human granulosa-lutein cells (hGLC) in vitro. Methods : hGLCs were isolated from patients undergoing IVF-ET cycles in which GnRH agonist or GnRH antagonist was used to prevent a midcycle gonadotropin surge. The cells were cultured for 3 days, and then treated with hCG 0.5, 1, and 10 IU/I, and insulin 0.01, 0.1, and 1 M in serum free conditions. In vitro P production was measured by enzyme immunoassay. Results : hCG stimulated P production by hGLCs from cycles in which GnRH antagonist was used, but a blunted response was seen in GnRH-agonist treated cycles. Insulin-stimulated P production was similar in cells from cycles in which GnRH-agonist or GnRH-antagonist treatment was used. Conclusions : Because insulin and hCG may share common pathways beyond the level of receptor activation, we hypothesize that GnRH agonist, but not GnRH antagonist, may affect the expression and/or activation of LH receptors in the hGLCs.     

The regional expression of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) in the placentae of women with pre-eclampsia

Insulin-like growth factors and their binding proteins regulate cellular proliferation, differentiation and function, and play an important role in placental development. IGF-II and IGFBP-1 are abundantly expressed by cells at the maternal-fetal interface and mediate cell-to-cell communication between trophoblasts and decidua. Placentae of pre-eclamptic pregnancies show villous cytotrophoblast proliferation, increased syncytial sprout formation and impaired trophoblast invasion. We hypothesized that the expression of IGF-II and IGFBP-1 by cells at the maternal-fetal interface is altered in pre-eclampsia. We determined the regional abundance and cellular localization of IGF-II mRNA and IGFBP-1 mRNA and protein in placentae from normotensive control and pre-eclamptic pregnancies. IGF-II mRNA was expressed in both the chorionic villi and basal plate decidua regions. Increased IGF-II mRNA abundance was observed in the intermediate trophoblasts of peri-infarct regions. IGFBP-1 expression was present only in the decidua of the basal plate and membranes, and this expression was decreased significantly in pre-eclamptic placentae. The increased IGF-II expression in the intermediate trophoblast surrounding placental infarcts suggests a role for IGF-II in placental repair or remodelling. Decreased IGFBP-1 mRNA expression in the basal plate decidua suggests that the increased concentrations of IGFBP-1 the circulation of pre-eclamptic women is not of decidual origin. The altered IGF-II and IGFBP-1 expression at the fetomaternal interface may be important in the pathophysiology of pre-eclampsia.