Enzymology of human myometrium: variations related to the hormonal milieu

The activities of various enzymes involved in glycolysis, tricarboxylic acid cycle and fatty acid oxidation were assayed in human myometrium. A gradient of the activities from fundal to cervical myometrium was observed. In contrast to studies performed in rodents, cyclic changes of glycolytic enzymes could not be detected. Hydroxyacyl-CoA-dehydrogenase (HAD) activity was higher in secretory phase myometrium and in cases with cystic hyperplasia of the endometrium than in proliferative phase myometrium. In pregnant myometrium, lactate dehydrogenase (LDH) and glycogen phosphorylase (GLP) were increased and in postmenopausal myometrium the activities of phosphofructokinase (PFK), LDH and succinate dehydrogenase (SDH) were decreased as compared to proliferative phase myometrium. We conclude that in the human myometrium, except for HAD, activities of enzymes involved in fuel metabolism are stable throughout the menstrual cycle and that only prolonged hormonal stimulation leads to alterations of some enzyme activities.     

The activities of some glycosaminoglycan-degrading enzymes in uterine leiomyomas

The activities of some glycosaminoglycan-degrading enzymes in uterine leiomyomas. Both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans (GAGs). In contrast to many normal and tumour tissues the amount of hyaluronic acid (HA) is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. We compared the activity of GAG-degrading enzymes in normal myometrium and in uterine leiomyomas. Growth of uterine leiomyomas results in significant reduction in the activities of neutral endoglycosidases degrading most of the sulphated glycosaminoglycans. The activities of acid endoglycosidases also decreased (with the exception of chondroitin-6-sulphate). Thus, the differentiated activity of glycosidases degrading glycosaminoglycans can be a factor modifying the quantity of GAGs.     

Cytochrome P-450 activity in human leiomyoma and normal myometrium

Variations in cytochrome P-450 levels may influence the responsiveness of uterine and breast tissue as well as carcinomas to endocrine therapy and may be of particular importance with agents such as tamoxifen (Nolvadex) where hydroxylation is known to alter therapeutic activities. Therefore, a sensitive spectrophotometric assay of cytochrome P-450 levels in reproductive tissue microsomes was developed to measure cyclohexane hydroxylase activity. Cyclohexane served as a substrate for several forms of cytochrome P-450. Human uterine leiomyomas (uterine fibroid tumor) contained significantly higher (p less than 0.01) cytochrome P-450 activity than adjacent normal myometrium. Specific activities for both leiomyomas (2.87 +/- 0.26 nmol/min/mg) and normal myometrium (1.60 +/- 0.11 nmol/min/mg) were in the range of those observed for untreated rabbit liver microsomes (1 to 3 nmol/min/mg). The contribution of smooth muscle in the specimen, the phase of the menstrual cycle, and the clinical diagnosis did not influence the level of cytochrome P-450 activity.     

Different expression of estrogen receptors alpha and beta in human myometrium and leiomyoma during the proliferative phase of the menstrual cycle and after GnRHa treatment.

Uterine leiomyomas (uterine fibroids) are sex-steroid dependent benign tumors. Limited knowledge is available regarding the role of estrogen and their receptors in the regulation of fibroids in premenopausal women, and in their shrinkage after treatment with a gonadotropin-releasing hormone analogue (GnRHa). The expression of the two subtypes of the estrogen receptor (ER), ER alpha and ER beta, was studied in leiomyoma and homologous myometrium from women in the proliferative phase of the menstrual cycle and from women treated with GnRHa. The mRNA levels of ER alpha and ER beta were monitored by solution hybridization and in situ hybridization, and receptor proteins were detected and localized by immunohistochemistry. Both ER alpha and ER beta were present in the leiomyomas and homologous myometrium. The ER alpha mRNA level in the leiomyomas was higher than in the surrounding myometrium. The ER beta mRNA level was lower than that of ER alpha in both groups. ER beta immunoreactivity was lower in leiomyomas when compared with the myometrium after GnRHa treatment, while ER alpha was higher in the leiomyomas. The present results imply that the increased ratio of ER alpha/ER beta observed in the fibroids after GnRHa treatment could reflect or be the cause of the shrinkage of the leiomyoma, which is the clinical outcome of this treatment.     

Estrogen and progesterone receptors in leiomyomas and normal uterine tissues during reproductive life

Estrogen and progesterone receptors (ER, PR) were measured in leiomyomas and normal uterine tissues. Estrogen receptors concentration was higher in endometrium than in leiomyomas, lowest in normal myometrium. In the case of progesterone receptors, the concentrations in endometrium and leiomyomas were similar whereas that of myometrium was lower. ER and PR concentration were similar in leiomyomas of the uterine fundus, body and isthmus and steroid receptor content in the inner parts of large myomas was the same as in the outer parts. ER and PR concentrations in tumor-bearing myometrium were not different from those in myometrium of a control group.     

Expression of basic fibroblast growth factor (bFGF), FGF receptor 1 and FGF receptor 2 in uterine leiomyomas and myometrium during the menstrual cycle, after menopause and GnRHa treatment

BACKGROUND: To investigate whether basic fibroblast growth factor (bFGF) is involved in the growth regulation of human uterine leiomyomas the expression of bFGF and its receptors was measured in leiomyomas and myometrium obtained under different endocrine conditions. METHODS: The expression of bFGF, fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor 2 (FGFR2) was analyzed by immunohistochemistry and Western blot. RESULTS: Twenty-seven women with leiomyomas included eight in the proliferative phase, seven in the secretory phase, six after menopause and six after GnRHa treatment. In the proliferative phase, bFGF staining in leiomyomas was significantly stronger than in any other leiomyoma group. After GnRHa treatment, the expression of bFGF in both leiomyomas and myometrium was weaker than in the proliferative phase. The staining of FGFR1 was less intense in proliferative phase myometrium than in myometrium from any other group, significantly weaker than in the secretory phase. The leiomyomas demonstrated homogeneous cytoplasmic FGFR1 staining that was similar in all groups, except in the GnRHa treated patients where a more intense staining was observed, significantly stronger than in proliferative phase leiomyomas. No tissue differences were observed for staining of FGFR2 and no significant differences were observed between the different groups. Slightly less staining of FGFR2 was found in leiomyomas in the secretory phase but it did not reach statistical significance. The specificity of immunostaining was confirmed by Western blot. CONCLUSIONS: We suggest that the regulation of bFGF, and to some extent also its receptors in leiomyomas and in myometrium, is influenced by sex steroid hormones. However, the lack of differences in expression between leiomyomas and myometrium favors the view that bFGF does not necessarily contribute to the differences in growth regulation in these tissues.     

Activity of matrix metalloproteinase-2 and -9 and contents of their tissue inhibitors in uterine leiomyoma and corresponding myometrium.

BACKGROUND AND AIM: Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are proteolytic enzymes degrading extracellular matrix proteins, mainly collagen type IV. Recent reports show that these proteases may be implicated in the growth of uterine leiomyoma. The aim of the present study was to evaluate the activity of MMP-2 and MMP-9, the contents of their tissue inhibitors (TIMP-1 and TIMP-2) and the immunolocalization of collagen type IV in uterine leiomyoma and corresponding myometrium. MATERIALS AND METHODS: Material for the study comprised specimens of uterine leiomyomas and corresponding myometrium derived from 20 hysterectomized women. The activity of MMP-2 and MMP-9 in tissue extracts was evaluated by semi-quantitative zymography. TIMPs were measured by enzyme-linked inmmunosorbent assay. Protein immunohistochemistry was applied for detection of collagen type IV. RESULTS: Activity and activation ratio of MMP-2 were significantly higher in leiomyomas than myometrium. The activity of MMP-9 was weak and did not differ between the investigated tissues. Contents of TIPM-1 and TIPM-2 were similar in both tissues. In both leiomyomas and myometrium, collagen type IV was localized in the extracellular matrix embedding bundles of smooth muscle cells, but was absent in areas of extracellular matrix accumulation within leiomyomas and in larger septa separating muscle fibers in normal myometrium. CONCLUSION: MMP-2 may be implicated in pathogenesis of leiomyoma.     

Apoptosis, cellular proliferation and expression of p53 in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

BACKGROUND: Cell proliferation, apoptosis and expression of p53 proteins were studied in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause. METHODS: Expression of ki-67 and p53 was analyzed by immunohistochemistry and by immunoblotting. Apoptosis was detected by in situ 3' end labelling of cells with DNA fragmentation. RESULTS: In both the proliferative and the secretory phases, ki-67 expression was higher in leiomyomas than in myometrium and both tissues showed higher expression in the secretory than in the proliferative phase. No difference in apoptotic index was observed between leiomyomas and myometrium or between the proliferative and secretory phases. After menopause, the expression of ki-67 as well as the apoptotic index was lower than in the proliferative and secretory phases and no significant difference between tissues was seen. Both leiomyomas and myometrium showed negative staining for p53. Immunohistochemical results regarding p53 were confirmed by Western blot. CONCLUSIONS: Our findings indicate that sex steroids influence the growth of leiomyomas by stimulating cell proliferation rather than by affecting apoptosis. The rate of cell proliferation is higher in fertile age than after menopause and appears to be enhanced under the influence of progesterone.     

Activity of matrix metalloproteinase-2 and -9 and contents of their tissue inhibitors in uterine leiomyoma and corresponding myometrium

Background and aim. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are proteolytic enzymes degrading extracellular matrix proteins, mainly collagen type IV. Recent reports show that these proteases may be implicated in the growth of uterine leiomyoma. The aim of the present study was to evaluate the activity of MMP-2 and MMP-9, the contents of their tissue inhibitors (TIMP-1 and TIMP-2) and the immunolocalization of collagen type IV in uterine leiomyoma and corresponding myometrium.

Materials and methods. Material for the study comprised specimens of uterine leiomyomas and corresponding myometrium derived from 20 hysterectomized women. The activity of MMP-2 and MMP-9 in tissue extracts was evaluated by semi-quantitative zymography. TIMPs were measured by enzyme-linked inmmunosorbent assay. Protein immunohistochemistry was applied for detection of collagen type IV.

Results. Activity and activation ratio of MMP-2 were significantly higher in leiomyomas than myometrium. The activity of MMP-9 was weak and did not differ between the investigated tissues. Contents of TIPM-1 and TIPM-2 were similar in both tissues. In both leiomyomas and myometrium, collagen type IV was localized in the extracellular matrix embedding bundles of smooth muscle cells, but was absent in areas of extracellular matrix accumulation within leiomyomas and in larger septa separating muscle fibers in normal myometrium.

Conclusion. MMP-2 may be implicated in pathogenesis of leiomyoma.     

Different expression of estrogen receptors α and β in human myometrium and leiomyoma during the proliferative phase of the menstrual cycle and after GnRHa treatment

Uterine leiomyomas (uterine fibroids) are sex-steroid dependent benign tumors. Limited knowledge is available regarding the role of estrogen and their receptors in the regulation of fibroids in premenopausal women ,and in their shrinkage after treatment with a gonadotropin-releasing hormone analogue (GnRHa). The expression of the two subtypes of the estrogen receptor (ER) ,ERα and ERβ, was studied in leiomyoma and homologous myometrium from women in the proliferative phase of the menstrual cycle and from women treated with GnRHa. The mRNA levels of ERα and ERβ were monitored by solution hybridization and in situ hybridization ,and receptor proteins were detected and localized by immuno-histochemistry. Both ERα and ERβ were present in the leiomyomas and homologous myometrium. The ERα mRNA level in the leiomyomas was higher than in the surrounding myometrium. The ERβ mRNA level was lower than that of ERα in both groups. ERβ immunoreactivity was lower in leiomyomas when compared with the myometrium after GnRHa treatment ,while ERα was higher in the leiomyomas. The present results imply that the increased ratio of ERα/ERβ observed in the fibroids after GnRHa treatment could reflect or be the cause of the shrinkage of the leiomyoma ,which is the clinical outcome of this treatment.     

Transforming growth factor beta and platelet-derived growth factor in human myometrium and in uterine leiomyomas at various stages of tumour growth

OBJECTIVE: Some authors suggest that growth factors are intermediate regulatory elements through which the ovarian hormones exert their growth-stimulatory effects on uterine leiomyomas. STUDY DESIGN: It was decided to compare the amounts of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) in myometrium and in uterine leiomyomas of various weights (small: less than 10 g and large: more than 100 g). The tissues were homogenised and extracted with 1M acetic acid or with 0.05 M Tris-HCl, pH 7.6. The extracts were assayed for TGF-beta and PDGF with the use of the ELISA technique. RESULTS: The Tris-HCl was more efficient at extracting solvent than 1M of acetic acid. Both myometrium and leiomyomas contained nanogram amounts of extractable TGF-beta and picogram amounts of PDGF. Western immunoblotting demonstrated that both factors exist as stable complexes, probably with extracellular matrix components. The PDGF/TGF-beta ratio in Tris-HCl extracts was higher in leiomyomas than in myometrium and it increased during tumour growth. CONCLUSION: It is known that low concentrations of TGF-beta induce proliferation of cells by stimulating autocrine PDGF secretion. Higher concentrations of TGF-beta1 evoke a reverse effect by the down-regulation of the PDGF receptor and by direct growth inhibition. The increase in the PDGF/TGF-beta ratio during tumour growth seems be important in tumour biology. The low amount of TGF-beta eliminates the inhibitory effect of this factor on cell proliferation and stimulates both autocrine PDGF secretion and promotes the synthesis of PDGF receptors. It is thus possible to bind more PDGF by myometrial cells resulting in a hyperplasia of myometrium and enhancement of extracellular matrix synthesis.     

Matrix metalloproteinases of human leiomyoma in various stages of tumor growth

The amounts and activities of matrix metalloproteinases (MMPs) were studied in human myometrium and uterine leiomyomas in various stages of growth. It was found that both myometrium and the investigated tumors contain collagenolytic enzymes. MMP-1, MMP-2, MMP-3 and MMP-9 were found. Gelatinase A (MMP-2) is the most abundant. In control myometrium only 10% of this enzyme exists in an active form, whereas in tumors, especially in large ones, the values reach 30%. It is suggested that the high activity of MMP-2 is responsible for remodelling of extracellular matrix in the growing tumors.     

子宫肌瘤细胞凋亡及增殖状况研究

目的 探讨细胞凋亡与凋亡调控基因在子宫肌瘤的发生、发展中的作用。方法 2000～2001年应用原位末端脱氧核苷酸转移酶标记(terminal deoxynucleotity transferase mediated dUTP nick end labeling,TUNEL)技术和免疫组织化学染色观察子宫肌瘤和正常子宫肌层组织中细胞凋亡和凋亡调控基因增殖细胞核抗原(PCNA的表达。结果 子宫肌瘤组织中细胞凋亡指数显著高于正常子宫肌层组织(P<0.01)。子宫肌瘤组织中PCNA蛋白阳性率高于正常子宫肌层组织(P<0.01)。凋亡细胞多分布在PCNA蛋白阴性区,阳性区仅有少量分布,且PCNA阴性区细胞凋亡指数高于阳性区。结论 细胞凋亡失控在子宫肌瘤生成过程中起重要作用,PCNA蛋白表达能比较准确地反映子宫平滑肌瘤的细胞增殖信息,子宫肌瘤的发生与增殖和(或)凋亡失衡密切相关。     

Magnetic resonance spectroscopy features of uterine leiomyomas

OBJECTIVE: The purpose of this study is to investigate the in vivo magnetic resonance spectroscopy features of uterine leiomyomas using long echo time and to characterize the spectral patterns of these lesions. METHODS: We calculated metabolites in 15 patients with uterine leiomyomas and myometrium of 20 healthy control subjects using single-voxel proton MR spectroscopy (point resolved spectroscopy technique, TE:136 ms). Voxels were placed at the center of the uterine leiomyomas. The peak areas of creatine, choline, lipid and lactate were determined. The MR spectroscopy results of uterine leiomyomas were compared with the spectroscopy results obtained from the myometrium of healthy control subjects. RESULTS: The characteristically obtained signal was choline, which was detected not only in 14 of the 15 leiomyomas (93.3%) but also in 18 of the 20 myometrium of control subjects (90%). The lipid signals were determined in 9 of 15 patients with uterine leiomyomas (60%) and 8 of 20 control subjects (40%). The lactate signal was obtained from six of 15 patients with leiomyomas (40%) but only two of myometrium (10%). The creatine signal was obtained from 4 of 15 patients with leiomyomas (26.6%) and 5 of 20 myometrium (25%). Among the tested parameters only lactate peak was statistically significant (p < 0.05). CONCLUSION: Proton MR spectroscopic imaging may be helpful for the investigation of the underlying pathophysiology of uterine leiomyomas. The presence of lactate and lipid signals in the spectrum may be a useful indicator of metabolic pathway of uterine leiomyomas.     

Specific binding sites for insulin in the human myometrium and leiomyomas of the uterus.

OBJECTIVE: To investigate whether there are increased binding sites for insulin in the leiomyomas of the uterus. STUDY DESIGN: Samples of myomas and myometrium were obtained from seven patients with myomas at the time of hysterectomy. Binding studies were performed with [125I] insulin. RESULTS: The percent total binding of [125I] insulin in the myomal tissues (mean +/- SE 20.7% +/- 2.3%/100 micrograms protein) was significantly higher (P less than 0.01) than that in the myometrium (14.7% +/- 1.3%/100 micrograms protein). Scatchard analysis revealed that the increase in binding is because of increase in receptor affinity. CONCLUSIONS: There is increased binding of insulin in the myomas because of increase in receptor affinity. This could increase the sensitivity of the biological response of the myomal cells to insulin.     

Comparison of intrauterine prostaglandin metabolism during pregnancy in man, sheep and guinea pig.

Metabolism of prostaglandin F2 alpha (PGF2 alpha) was studied in uterine tissues from pregnant women (n = 10), sheep (n = 6) and guinea pigs (n = 6). Two maternal tissues, myometrium and decidua or maternal placenta, and two fetal tissues, fetal placenta and membranes, were studied in each species. PGF2 alpha was metabolized via the well-known pathway into 15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha, while 13,14-dihydro-PGF2 alpha was tentatively identified in some tissues. The presence of 15-hydroxyprostaglandin dehydrogenase (PGDH) and 13,14-prostaglandin reductase was demonstrated in all tissues from each of the three species studied, but the quantitative intrauterine distribution of these enzymes differed from one species to another. In man, PGDH activities were highest in fetal membranes followed by fetal placenta and then by decidua and myometrium. In sheep, PGDH activities were highest in the maternal placenta followed in decreasing order by myometrium, fetal placenta and membranes. In the guinea pig, PGDH activity was highest in the fetal membranes followed in decreasing order by maternal placenta, fetal placenta and myometrium. Quantitative assay of PGDH showed that PGDH activity in the pregnant uterus is higher in women than in sheep and guinea pig. The results are discussed in relation to the involvement of prostaglandins in parturition.     

Expression of von Willebrand's factor,CD34, CD31, and vascular endothelial growth factor in uterine leiomyomas

OBJECTIVE: To compare the vascular parameters of uterine leiomyomas and normal myometrium, to correlate these parameters with vascular endothelial growth factor (VEGF) expression and clinical/pathological parameters, and to compare vascular parameters according to the endothelial markers used.DESIGN: An immunohistochemical technique was applied to formalin-fixed paraffin-embedded tissue samples, using antibodies against von Willebrand's factor (FvW), CD34, CD31, and VEGF. The intratumoral vascular area (VA), microvessel density (MVD), and vascular luminal area (VLA) were determined with an image analyser.SETTING: University teaching hospital.PATIENT(S): Thirty-two patients with uterine leiomyomas underwent conservative surgery. Twenty leiomyoma-free patients undergoing hysterectomy were the controls.INTERVENTION(S): Immunohistochemical and morphometrical analysis.MAIN OUTCOME MEASURE(S): Measurements of VA, MVD, and VLA.RESULT(S): The CD34 labeling showed decreased VA in myomas compared with myometrium. Decreased MVD and an increased VLA in myomas were found with FvW and CD34 labeling. The VA, MVD, and VLA were not related to VEGF expression or to clinical/pathological parameters. Similar results for VA and MVD were obtained with FvW and CD34 labeling.CONCLUSION(S): Leiomyomas have a smaller vascular area, a lower microvessel density, and a higher vascular luminal area than normal myometrium.     

Gene expression and tyrosine kinase activity of insulin receptor in uterine leiomyoma and matched myometrium

Objective Our objective was to determine the insulin receptor (IR) mRNA levels and IR tyrosine kinase activity in normal myometrium and leiomyoma.Design Experimental study.Setting Academic research center.Patients Five women with leiomyoma submitted to hysterectomy.Intervention Plasma membrane fraction of human myometrium and leiomyoma were prepared and samples were incubated with and without insulin. mRNA was isolated and RT-PCR with specific primers was performed.Main outcome measure Western blots of plasma membranes incubated with and without insulin were performed. Chemoluminescent methods followed by densitometry were used to assess IR autophosphorylation. RT-PCR with specific primers for IR gene sequences was used to determine IR mRNA levels.Results IR mRNA levels in myometrium (0.634±0.038) and in leiomyoma (0.649±0.047; p=0.813) were not different. The degree of insulin-stimulated IR autophosphorylation (relative optical density of the 95 kDa band) was also not different between myometrium (1.496±0.310) and leiomyoma (1.593±0.129; p=0.650).Conclusions There was no difference in IR receptor expression and IR autophosphorylation between normal myometrium and leiomyoma. Other steps of insulin signaling chain may participate in the altered proliferation of leiomyomas.     

Onapristone suppresses prolactin production in explant cultures of leiomyoma.

OBJECTIVE: To assess the action of onapristone, a type I antiprogestin, on prolactin (PRL) production by explant cultures of leiomyoma and myometrium. DESIGN: Explant cultures of myometrium and leiomyomas from 3 premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle. MAIN OUTCOME MEASURES: PRL secretion measured by radioimmunoassay. RESULTS: PRL secretion was decreased in leiomyomas by onapristone. There was no effect in the myometrium. There was no additional effect with the addition of the type II antiprogestin mifepristone (RU 486). CONCLUSION: PRL production is suppressed in leiomyomas but not in myometrium after treatment with onapristone in vitro. This suppression may serve as a marker for the clinical effectiveness of agents used in the treatment of leiomyomas.