The content of RNA and DNA in human myometrium and fibroids obtained at different endocrine conditions varied, with the highest RNA/DNA ratio in tissues from pregnant patients, intermediate ratios in women during the menstrual cycle and the lowest in tissues from postmenopausal and GnRHa-treated patients. mRNA expression of two house-keeping genes, gamma-actin and GAPDH, was highest in myometrium from pregnant women, intermediate in untreated women of fertile age and lowest in tissues from GnRHa-treated and postmenopausal women. To control for degradation of nucleic acids when measuring mRNA expression, we suggest additional analysis of gene(s), where the expression pattern is known, and that expression, whenever possible, is related to DNA, which is a more stable parameter than RNA and total nucleic acids, when there are differences in proliferation between tissues and/or groups of patients.

73 Complete hydatidiform mole 241 Computerized cardiotocography 37 Congenital factor X deficiency 114 Conservative treatment 211 Cumulative recurrence hazard 14 Cytokine(s) 68, 189

The development and functions of female reproductive tissues are regulated by the actions of two major sex steroid hormones, estrogen and progesterone. To investigate estrogen-dependent gene expression in the rat uterus, we studied the effect of ovariectomy with or without estrogen treatment on the uterine expression of 3000 genes using cDNA microarrays. Many genes were regulated by either treatment, but only few were reciprocally regulated by these contrasting treatments. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include follistatin-related protein, Thy-1 glycoprotein, alpha-fodrin, CD24, immediate early response 5, insulin-like growth factor-binding protein 2, growth response protein CL-6 (INSIG-1), ladinin1, class I major histocompatibility complex heavy chain, lactadherin, ezrin, and Fas-activated serine/threonine kinase. Because of their function as regulators of proliferation and apoptosis, CD24, insulin-like growth factor-binding protein 2, and Fas/Fas ligand were examined further by immunohistochemical expression and tissue localization analysis. Our analysis confirms a contrasting regulation of these gene products by ovariectomy and estrogen treatment. The present study identifies novel mediators of estrogen actions in the uterus and provides genome-wide expression data from which novel hypotheses regarding uterine function can be generated.

Objective: To determine mRNA and protein expression of the progesterone receptor (PR) and insulin-like growth factor-I (IGF-I) in myometrium and fibroids.Design: Retrospective clinical study.Setting: Hospital-based and university-affiliated research laboratories.Patient(s): Twelve women in the proliferative phase and six women treated with GnRH analogue (GnRH-a).Intervention(s): Blood sampling and collection of myometrium and fibroids.Main Outcome Measure(s): PR and IGF-I mRNA levels in fibroids and myometrium were analyzed by solution hybridization and in situ hybridization whereas the proteins were localized by immunohistochemistry.Result(s): Fibroids and myometrium from women in the proliferative phase showed significantly higher PR mRNA than the corresponding tissues from GnRH-a-treated women. The amount of cells positively stained for PR-AB and PR-B in fibroids and myometrium decreased after GnRH-a treatment compared with in the proliferative phase. The IGF-I mRNA in both fibroids and myometrium in the proliferative phase was significantly higher than those after GnRH-a treatment. The immunostaining of IGF-I showed no difference between the two tissues. There was weaker immunostaining in the GnRH-a-treated group compared with in the proliferative phase group.Conclusion(s): The shrinkage of fibroids after steroid deprivation is associated with alterations in PR and IGF-I expression.

We used a PCR-based subtraction method, representational difference analysis of cDNA (cDNA--RDA), to identify genes with a higher expression in leiomyomas in comparison with the corresponding myometrium during the proliferative phase of the menstrual cycle. Increased expression of the genes for pregnancy-associated plasma protein A (PAPPA), tomoregulin, cellular retinoid acid binding protein 1 (CRABP1), zinc finger protein 185 (ZFP 185) and latent transforming growth factor beta binding protein 2 (LTBP2) was demonstrated in individual leiomyoma samples compared with corresponding myometrium. Additionally, a specific positive immunostaining of LTBP2 was found in the smooth muscle cells of both leiomyomas and myometrium. These genes may be part of previously unidentified molecular mechanisms responsible for the selective growth advantage of leiomyomas compared with myometrium. This work expands our knowledge about the molecular nature of leiomyomas and provides novel candidate genes to further explore in relation to their function during leiomyoma growth.

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause. The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.

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.

The acute toxicity of diethylnitrosamine (DEN) to the liver has been well documented in the literature, but whether DEN also affects the endocrine parameters has been addressed in only a few studies. We thus investigated the effects of DEN on pituitary, serum hormone levels, and certain sex-differentiated liver enzymes in this study. Adult male Wister rats were intraperitoneally injected with DEN at a single dose of 200 mg/kg and were sacrificed at 1, 3, 7, and 35 days after injection; DEN-treated females were included as controls at days 7 and 35. Electron microscopic observation showed that during the first week after injection, all types of granular cells of the anterior pituitary in male animals exhibited cellular damage, including disrupted organelles and cellular structure, as well as pyknotic or lytic nuclei. Many undamaged secretory cells exhibited dilated endoplasmic reticula, hypertrophic Golgi complexes, and peripheral location of secretory granules, which usually are morphologic features of increased cellular activities. In male rats, the serum level of total testosterone decreased and the corticosterone increased 1 day after DEN treatment. The serum level of growth hormone (GH) decreased and the prolactin level increased on day 3. The hepatic expression of the male-specific cytochrome P450 2C11 (CYP2C11) decreased to 1-5% of the normal levels during the first week and was still 50% lower than the normal level on day 35, whereas the female-specific CYP2C12 expression increased only slightly. Activities of the male predominant 16alpha, 16beta, and 6beta hydroxylation of androstenedione by microsome decreased in an in vitro assay, whereas the non-sex-differentiated 7alpha hydroxylation and the female-predominant 5alpha reduction of androstenedione were unaffected. In female rats, decreased serum GH level was observed on day 7. The CYP2C12 expression in females was decreased to about 1% and 80% of the normal levels on day 7 and day 35, respectively, but the CYP2C11 expression was unchanged. These data suggest that in male rats, DEN treatment may cause pituitary damage, disturb serum hormone levels, and induce long-lasting reduction of sexual dimorphism in certain liver functions.

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.

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. Note

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.

Sex steroids influence the growth of mammalian uterine tissues and the proto-oncogenes c-fos and c-jun have been implicated in the cascade of cellular events induced by the cyclic influence of oestrogen and progesterone. To investigate the role of these proto-oncogenes for fibroid growth their mRNA expression was measured in myometrium and fibroids under different hormonal conditions, using a solution hybridization method. Fibroids and myometrium were collected at surgery from premenopausal, postmenopausal and pregnant women as well as women treated with a gonadotrophin releasing hormone agonist (GnRHa; Goserelin). The phase of the menstrual cycle was determined in all the untreated, premenopausal, non-pregnant women. The mRNA expression of c-fos and c-jun in fibroids was significantly lower than in homologous myometrium. No significant differences in c-fos expression were observed in myometrium, or fibroids, due to menstrual cycle phase, GnRHa treatment, pregnancy or the menopause. The c-jun expression in myometrium from pregnant women without fibroid disease was significantly higher than the corresponding control myometrium from premenopausal, non-pregnant women. These results demonstrate a tissue difference in the expression of c-fos and c-jun between myometrium and fibroids.

Thioredoxin is a small multifunctional protein which acts as a dithiol hydrogen donor for ribonucleotide reductase in DNA synthesis. Thioredoxin participates in the regulation of different metabolic processes, such as changes in the activity of different enzymes, receptors or transcription factors. The aim of the present study was to determine possible differences in the expression of thioredoxin between myometrium and fibroids in women during different periods of life. Thioredoxin mRNA concentrations were determined in myometrial and fibroid tissues obtained from women during the menstrual cycle, during treatment with an analogue of gonadotrophin releasing hormone (GnRH agonist), in the postmenopausal period (PMP) and during pregnancy. The concentration of thioredoxin mRNA was measured by a solution hybridization method. The localization of thioredoxin protein was examined by immunohistochemistry. There were significantly lower levels of thioredoxin expression in both fibroids and myometrium from GnRH agonist treated and PMP women in comparison with the pregnant women. No difference in thioredoxin expression was found between myometrium and fibroids from the same woman or between myometria from uteri with or without fibroids in the same patient group. Thioredoxin expression in uterine fibroids does not seem to be up-regulated, but changes in response to the endocrine conditions in a similar way to that observed in the myometrium.

The expression of insulin-like growth factor-I (IGF-I) was measured at the mRNA and protein level in myometrium and fibroids from women with and without preoperative treatment with a gonadotrophin-releasing hormone (GnRH) agonist for 3 months, from post-menopausal women, from pregnant women and in myometrium from women without fibroid disease. Women with menstrual periods were classified according to the phase of the cycle. In tissues from non-treated premenopausal women, IGF-I mRNA expression was significantly higher in fibroids than in myometrium, with no differences related to phase of the menstrual cycle. In post-menopausal women and in GnRH agonist-treated women responding to treatment, similar mRNA expression was seen in myometrium and fibroids but the concentrations were lower than in untreated premenopausal women. The IGF-I mRNA value in fibroids from pregnant women was higher than in any other group and myometrium from pregnant women exhibited higher mRNA expression than myometrium from non-treated premenopausal women. The IGF-I protein was more abundant in fibroids than in myometrium of non-treated premenopausal and of pregnant women and in both tissues the concentration was significantly higher in the group of pregnant women. The IGF-I protein concentrations in fibroids and myometrium from GnRH agonist-treated and post-menopausal women were similar to those from premenopausal non-treated women. High sex steroid concentrations in pregnant and non-pregnant women of fertile age seem to be associated with a higher expression of IGF-I in fibroids than in myometrium, suggesting that IGF-I contributes to the selective growth advantage of these tumours.