BACKGROUND:Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF is a stromally derived, paracrine growth factor specifically mitogenic for a variety of epithelial cells. The KGF receptor (KGFR), which is a splice variant of the FGF receptor-2 (FGFR-2)/bek gene, is expressed only in epithelial cells. In this study, the expression of mRNAs encoding KGF, KGFR, and FGFR-2 in endometrial adenocarcinoma and in carcinosarcoma tissues was examined and the expression of the same mRNAs was compared with cycling endometrium. METHODS:Specimens of tumor tissue were collected from 14 women with well differentiated endometrial adenocarcinoma and from 4 women with carcinosarcoma. All samples were obtained at the primary surgery before any treatment was initiated. In addition, endometrial tissues from 19 premenopausal women with normal menstrual cycles were examined. The expression of specific mRNAs in the endometrial samples was assessed using quantitative reverse transcriptase polymerase chain reaction. The results were analyzed by the nonparametric Kruskal-Wallis statistic. RESULTS:The KGF mRNA expression was significantly lower in endometrial adenocarcinoma tissue compared with cycling endometrial tissues, whereas no difference was found between carcinosarcoma tissue and cycling endometrium. The relative level of KGFR mRNA in endometrial adenocarcinoma did not differ from that in cycling endometrium, but was significantly higher compared with carcinosarcomas. No differences were observed in FGFR-2 mRNA expression between cycling endometrium and tumor tissues. CONCLUSIONS:To the authors' knowledge, this study demonstrates for the first time the expression of KGF, KGFR, and FGFR-2 mRNAs in endometrial adenocarcinoma and in carcinosarcoma tissues. The relative level of KGF mRNA expression in adenocarcinoma tissue is decreased compared with that in cycling endometrium. The change in epithelial/stromal cell prominence between cycling endometrium and adenocarcinoma tissue may account for the difference in KGF expression but does not explain why KGF receptor expression in same tissues remains unchanged. The impact of altered KGF expression for endometrial tumorigenesis is still unknown.

OBJECTIVE:Our purpose was to evaluate the relationship of obesity, hyperinsulinemia, and hyperandrogenemia on serum insulin-like growth factor binding protein-1 levels in women with the polycystic ovarian syndrome.STUDY DESIGN:Insulin-like growth factor binding protein-1, insulin, and androgen levels were studied during a 3-hour intravenous glucose tolerance test in 16 women with polycystic ovarian syndrome (nine obese and seven nonobese) and 20 healthy control women (10 obese and 10 nonobese).RESULTS:Positive correlations were observed between basal (r = 0.77, p = 0.04) and area under curve (r = 0.86, p < 0.001) insulin-like growth factor binding protein-1 with basal androstenedione in the nonobese women with polycystic ovarian syndrome but not in other groups of women examined or between other androgens and insulin-like growth factor binding protein-1. An inverse relationship was observed between log area under curve insulin-like growth factor binding protein-1 levels and log body mass index in polycystic ovarian syndrome (r = -0.54, p = 0.03) and in normal women (r = -0.43, p = 0.06). The log area under curve insulin-like growth factor binding protein-1 level was approximately an inverse linear function of log area under curve insulin response for both women with polycystic ovarian syndrome (r = -0.70, p < 0.001) and control women (r = -0.72, p < 0.001). Additionally, after the area under curve insulin response during intravenous glucose tolerance testing was controlled for, the decline in area under curve insulin-like growth factor binding protein-1 responses was on average 66% less in both obese and nonobese women with polycystic ovarian syndrome compared with same-weight controls (95% confidence interval 110% to 270%, p = 0.04).CONCLUSION:These data indicate that insulin and body mass index are the major determinants of circulating insulin-like growth factor binding protein-1 and that chronic hyperandrogenemia does not appear to further reduce serum insulin-like growth factor binding protein-1 levels in obese or normal-weight women with polycystic ovarian syndrome.

In this study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to compare the expression of mRNAs encoding endothelin-1 (ET-1), endothelin receptors type A (ETA-R) and type B (ETB-R) and ET-1-degrading enzyme neutral endopeptidase 24.11 (NEP) in 15 endometrial cancer tissues and 13 normal endometrial tissues. The relative levels of ET-1 mRNA in endometrial cancer tissues did not differ from those in normal endometrium. Both ETA-R and ETB-R mRNA levels were significantly lower in endometrial cancer tissue than in normal endometrium (P < 0.001). The complete lack of NEP mRNA in endometrial cancer tissues was in marked contrast to results from normal endometrium (P < 0.001). In conclusion, differential expression of mRNAs encoding ET-R and NEP in normal endometrium and endometrial cancer suggests that ET action is altered in endometrial cancer compared with normal endometrium.

Background . The aim of the present study was to evaluate the expression of mRNA for the keratinocyte growth factor and the keratinocyte growth factor receptor in human endometrium at different stages of the menstrual cycle. The role of estrogen and progesterone in regulating the expression of the mRNAs encoding keratinocyte growth factor and its receptor was further examined by studying the effect of continuous progestin (endometrium exposed to levonorgestrel releasing intrauterine contraceptive device), and continuous estrogen (endometrium hyperplasia) on the endometrium. Methods . The expression of mRNA in endometrial samples was evaluated using reverse transcriptase polymerase chain reaction. Results . The expression of KGF mRNA was found to vary during the menstrual cycle, with the highest levels in the progesterone‐dominated late‐secretory stage endometrium. Keratinocyte growth factor mRNA expression was low in both the endometrium that had been under the influence of continuous progestin (atrophic endometrium) and continuous estrogen (hyperplastic endometrium). The highest level of keratinocyte growth factor receptor mRNA expression was seen in late‐proliferative stage of the menstrual cycle and in hyperplasia when the estrogen exposure to endometrium is high. A low receptor mRNA level was found in endometrium exposed to continuous progestin. Conclusion . The results suggest that keratinocyte growth factor mRNA expression is progesterone dependent, whereas keratinocyte growth factor receptor mRNA expression seems to be more estrogen than progesterone dependent.

Knip M, Tapanainen P, Pekonen F, Blum WF. Insulin-like growth factor binding proteins in prepubertal children with insulin-dependent diabetes mellitus. Eur J Endocrinol 1995:133:440–4. ISSN 0804–4643 To study the possible role of insulin-like growth factor binding proteins (IGFBPs) in the discrepancy between normal or only slightly retarded growth and substantially reduced concentrations of insulin-like growth factor I (IGF-I) in prepubertal children with insulin-dependent diabetes mellitus (IDDM), we measured the plasma concentrations of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-3 and free insulin in 24 prepubertal diabetic subjects and 12 control children. In addition, the growth hormone response to exercise was evaluated. The diabetic children had significantly decreased peripheral IGF-I levels (8.2 + 1.1 (sem) vs 16.7 + 2.5 nmol/l; p < 0.001), whereas the concentrations of free insulin were increased (217 + 14 vs 103 + 21 pmol/l; p < 0.001). The concentrations of IGFBP-1 and IGFBP-3 were of the same magnitude in both groups. The diabetic children had significantly increased levels of IGFBP-2 (465 + 13 vs 416 + 14 μg/l; p = 0.029), which were inversely related to the circulating IGF-I levels (r = −0.35; p = 0.034). The diabetic and control children had comparable growth hormone responses to exercise. Diabetic children with poor glucose control had even lower IGF-I levels than those with moderate metabolic control (6.0 + 0.8 vs 10.3 + 1.7 nmol/l; p = 0.037). No differences could be observed in the plasma concentrations of various IGFBPs between these two groups of diabetic subjects. The absence in prepubertal diabetic children of increased IGFBP-1 levels observed in adolescent and adult patients with IDDM may contribute to their maintained linear growth, despite definitely decreased IGF-I concentrations. The role of increased IGFBP-2 levels in prepubertal children with IDDM remains open, but the inverse relationship between IGF-I levels and IGFBP-2 concentrations suggests that IGF-I may be involved in the regulation of IGFBP-2. Mikael Knip, Department of Pediatrics, University of Oulu, FIN-90220 Oulu, Finland

The expression of mRNAs encoding endothelin-1 (ET-1) and its receptors (ETA-R and ETB-R) as well as the ET degrading enzyme, neutral endopeptidase 24.11 (NEP), was determined in tissue samples of endometrium, myometrium and leiomyoma by using a reverse transcriptase polymerase chain reaction (RT-PCR) technique. ET-1 mRNA was detected in all samples studied. The level of ET-1 mRNA was higher in endometrium than in myometrium (p < 0.01) and leiomyoma (p < 0.001). The ETA-R mRNA was more abundant in endometrium than in myometrium (p < 0.001). For ETB-R mRNA there was no difference between these tissues. In contrast to ETA-R mRNA, which was more abundant in leiomyoma than in myometrium (p < 0.01), the ETB-R mRNA was less abundant in leiomyoma (p < 0.01). The NEP mRNA was detected in all endometrial samples but not in myometrium and leiomyoma. Our results show that the expression and relative levels of mRNAs encoding ET-1, ETA-R, ETB-R, and NEP vary in different tissue compartments of the human uterus. Since the net biological action of ET-1 in a particular cell type presumably depends on the balance between the peptide itself, its receptors and degrading enzymes, these results suggest different roles for ET-1 action in uterine endometrium, myometrium and leiomyoma. The difference in relative abundance of ETA-R and ETB-R mRNAs between myometrium and leiomyoma suggests that an altered ET-R gene expression may be a contributing factor in myomal growth.

The expression of epidermal growth factor receptor (EGFR) in breast cancer patients was correlated to the disease-free survival. The study included 149 breast cancer patients. The mean follow-up time was 4.2 years. There was an inverse correlation between EGFR and estrogen receptor (p < 0.003) and progesterone receptor (p < 0.013). Subdivision of EGFR showed that patients with EGFR > or = 0.60%, regarded as EGFR positive, had a worse prognosis than patients with EGFR binding < 0.60%, regarded as EGFR negative (p = 0.004). In the ER negative group, EGFR positive patients had a shorter relapse-free time than patients with EGFR negative cancer (p < 0.009). The same subdivision among ER positive patients showed no statistically significant difference.

The insulin-like growth factor-I is an important mitogen and has a growth promoting property, especially in breast cancer. This work analyses the prognostic value of the insulin-like growth factor receptor-I (IGFR-I), which belongs to the group of membrane receptors for growth factors. The study included 126 patients. 49 patients (39%) were IGFR positive (≥4.0%). There was a significant correlation between IGFR and oestrogen receptor (ER) status (P = 0.001), but not between IGFR and progesterone receptor status (PR; P = 0.07). There was no correlation between node status and IGFR. The expression of IGFR had a strong significance in the disease-free analysis (P = 0.0108). The IGFR status was not of predictive value in the node-negative subgroup (64 patients). Within the ER-negative group, the disease-free analysis further stratified with IGFR revealed that patients with IGFR-positive and ER-negative cancers are in a worse situation than IGFR-negative ER-negative cancer patients (P = 0.01).

The insulin-like growth factor (IGF) system is thought to function as a mediator of steroid hormone actions in the endometrium. IGFs (IGF-I and IGF-II) are also potent mitogens in endometrial cancer. The biological actions of IGFs are modulated by specific binding proteins (IGFBP)--6 cloned and sequenced so far--which may either inhibit or enhance the effects of IGF at the cellular level. In the endometrium, IGFBP-1 gene expression is stimulated by progesterone and inhibited by insulin, while IGFBP-1 inhibits the mitogenic action of IGF-I. In this study, we used a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to investigate IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 and IGFBP-6 gene expression in endometrial cancer tissues. Endometrial cancer tissue samples were collected from 20 women (aged 54-79 yrs) with stage I to II well-differentiated endometrial adenocarcinoma. Samples of normal endometrium (n = 14) obtained from women undergoing tubal ligation in various phases of the menstrual cycle, and normal early-pregnancy endometrium (decidua) were studied for comparison. In endometrial cancer tissues, the IGFBP-1 mRNA was undetectable or minimally expressed when studied by RT-PCR. The mean (+ SD) levels of IGFBP-2 and IGFBP-4 and IGFBP-5 mRNAs in endometrial cancer tissues did not differ from those in normal endometrium, in which no cyclic variation was observed, suggesting that the genes encoding IGFBP-2, IGFBP-4 and IGFBP-5 are not hormonally regulated in the endometrium. The IGFBP-6 mRNA expression showed a significant cyclic variation in normal endometrium, with low levels in late-proliferative and early- to mid-secretory phases and high expression in late-secretory and early-proliferative phases. In endometrial cancer tissues, the mean IGFBP-6 mRNA level was similar to that in cycling endometrium during the peri-ovulatory period. In summary, a continuous stimulation of the endometrial epithelial cells by IGFs with suppressed IGFBP-1 expression may lead to an imbalance in the IGF system of the endometrium and trigger an uncontrolled cell proliferation, ultimately resulting in malignant transformation.

The expression of insulin-like growth factors and their binding proteins in normal human peripheral lymphocytes was studied using the reverse transcriptase polymerase chain reaction method and Western ligand blotting. A quantitation of RT-PCR products was used to study the differences between normal and PHA stimulated lymphocytes. Normal freshly collected lymphocytes expressed mRNAs for both IGF-I receptor and IGF-II receptor but no expression of the corresponding growth factors was detectable. After stimulation with phytohemagglutinin the lymphocytes, however, expressed both IGF-I and IGF-II. Of the five IGFBPs examined, unstimulated lymphocytes expressed only IGFBP-2 and -3. Stimulated lymphocytes expressed IGFBP-4 and -5, in addition to IGFBP-2 and -3, whereas IGFBP-1 mRNA remained undetectable. The ligand blotting of lymphocyte conditioned media revealed production of 34K, 43K and 49K IGFBPs. The addition of estrogen, progesterone, IGF-I or growth hormone did not affect secretion of IGFBPs by lymphocytes.

Evidence available supports the concept that the proliferative response of the uterus to oestrogen is through activation of local regulatory factors, IGFs amongst them. Oestrogen stimulates the expression of IGF-I and IGF-I receptor mRNAs, whereas progesterone induces the major endometrial IGFBP, IGFBP-1, which is proposed to act as an inhibitor of IGF actions in normal endometrium. Since both benign and malignant uterine diseases are related to oestrogen action, the hormonally regulated local IGF system may play an important role in the pathophysiology of uterine diseases. In support of this hypothesis, we found differences in the expression of IGFBP mRNAs and in the presence of translated IGFBPs between benign and malignant uterine tissues. Furthermore, receptor binding of IGF-I has been found to be higher in neoplastic uterine tissues compared to normal tissues.

To improve the quality of life among women during the three decades following the menopause, every effort should be made to prevent the adverse effects of ageing. Broad joint initiatives, involving various disciplines such as, cardiology, urology, oncology, orthopaedics and gynaecology, are needed to optimise medical care during the climacteric. Oestrogen-progesterone treatment is the most important means currently available for the prevention of postmenopausal problems. This form of hormone therapy also appears to provide protection against osteoporosis and cardiovascular disease.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a major protein in amniotic fluid. In this study, we evaluated the diagnostic potential of IGFBP-1 measurement in cervical/vaginal secretions as an indicator of ruptured fetal membranes. Data were also compared with those obtained by the ROM-check Membrane Immunoassay (Adeza Biochemical, Sunnyvale, California) that is based on the detecton of fetal fibronectin in vaginal fluid. In women with intact membranes and not in labor, the concentration of IGFBP-1 in specimens obtained from the cervix and immersed in 0.5 ml of assay buffer ranged from <0.5 to 90 μg/l, whereas in specimens obtained less than 8 h after spontaneous or artificial rupture of membranes it varied between 175 and 20,000 μg/l, the median being 1,900 μg/l. The values greater than 100 μg/l were interpreted as containing amniotic fluid. The IGFBP-1 measurement and the ROM-check Membrane Immunoassay were carried out parallel in the vaginal swab specimens obtained from 54 pregnant women from 1 h to 1 week after the rupture. Twenty-four women had ROM confirmed from 1 h to 1 week earlier. In this group of patients, IGFBP-1 concentration 100μ had a sensitivity of 75% and a specificity of 97% in diagnosis of ROM. The corresponding numbers for a positive ROM-check were 92% and 80%. The positive predictive value was 95% for the IGFBP-1 measurement compared to 79% for the ROM-check test. These data show that IGFBP-1 is an ideal marker for amniotic fluid and rapid tests for its measurement in vaginal fluid might provide a reliable tool for detection of ruptured fetal membranes, if not more than 12 h have elapsed since the leakage of amniotic fluid into the vagina.

The expression of mRNAs for keratinocyte growth factor (KGF) (also called FGF-7) and its receptor was evaluated in normal human endometrium and myometrium as well as in myoma and in endometrial adenocarcinoma cell lines using reverse transcriptase polymerase chain reaction. Both KGF and its receptor mRNA are expressed in the human endometrium throughout the menstrual cycle, whereas fibroblast growth factor receptor 2 (FGFR-2) mRNA expression is low in this tissue. In endometrial stromal cell enriched preparations KGF mRNA dominates with little expression of KGF receptor (KGFR) and FGFR-2, whereas in the epithelial cell-enriched fraction the KGFR mRNA dominates. Human myometrium and myoma express mRNA for KGF, but not for KGFR. FGFR-2 is expressed in both myometrial and myoma tissues. None of the five endometrial adenocarcinoma cell lines studied expressed KGF mRNA, whereas all cell lines expressed mRNA for either KGFR or FGFR-2 or for both receptors. The results show a selective expression of KGFR and the closely related FGFR-2 in the human uterus with the former being expressed in the endometrium and the latter predominantly in the adjacent myometrium. In the endometrial tissue, selective expression of KGF in stromal cells and KGFR in epithelial cells supports the paracrine function of KGF in epithelial tissue.

The effect of local intrauterine progestin on endometrial insulin-like growth factor-binding protein-1 (IGFBP-1) production was studied in 60 women using a levonorgestrel-releasing intrauterine device (IUD). Intrauterine progestin was a potent stimulator of stromal cell IGFBP-1 production, with 97% of endometrial specimens showing positive staining by immunohistological methods. After 5 yr or more of intrauterine progestin exposure, 100% (n = 20) of the tissues remained strongly positive for IGFBP-1. The IGFBP-1 content in endometrial tissue homogenates reached values as high as 10 micrograms/mg protein when measured by immunoradiometric assay. In contrast to the continuous endometrial IGFBP-1 production induced by local progestin, no such effect could be found in endometria from subjects with sc progestin-releasing implants or copper IUDs. Although the levonorgestrel-releasing IUD had a striking effect on local endometrial IGFBP-1 production, it had no effect on serum IGFBP-1 levels. By Western ligand blot analysis, the domainating IGF-binding species in endometria exposed to intrauterine progestin was of 28K mol wt, corresponding to IGFBP-1, whereas no IGFBP species of 31-43K, corresponding to IGFBP-2 or IGFBP-3, were detected.

The effect of ovulation induction on serum insulin-like growth factor binding protein 1 (IGFBP-1) level in relation to sex hormone binding globulin (SHBG) levels was evaluated. Serum samples were collected 8 to 12 days after ovulation from 26 women undergoing ovulation induction with clomiphene citrate (CC), and from 58 women treated with CC in combination with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG). In addition, serum samples were obtained from 63 spontaneously ovulating women and from 12 women during an anovulatory cycle. Luteal phase serum IGFBP-1 levels were 4.22 +/- 2.95 micrograms/L (P less than .05) in the CC group and 7.31 +/- 6.13 micrograms/L (P less than .001) in the CC/hMG/hCG group as compared to unstimulated ovulatory cycles (2.64 +/- 2.52 micrograms/L). No significant difference in IGFBP-1 levels was seen between spontaneously ovulatory and anovulatory cycles. The serum IGFBP-1 levels correlated positively to SHBG levels (r = .52, P less than .001). The data show that ovulation induction increases serum IGFBP-1 levels in parallel to SHBG levels, indicating that ovarian stimulation, which results in increased steroid hormone production, also induces changes in other factors known to modulate steroid hormone actions.

Breast tumor cell lines have been shown to secrete at least five distinct insulin-like growth factor (IGF) binding proteins (IGFBP), the secretion being related to the estrogen receptor (ER) content. In this study we investigated IGFBP mRNA expression and IGFBP content in relation to ER content in human breast tumors. Tissue specimens from 47 breast cancers were studied. In five cases the adjacent histologically normal tissue was also analyzed. IGFBP content in tissue homogenates was studied by Western ligand blot analysis, using [125I] IGF-I as a label, and IGFBP mRNA expression by reverse transcriptase polymerase chain reaction. The results show that human breast tumors express mRNAs encoding IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5. The pattern of IGFBPs in different tumors varies. No correlation exists between ER content and IGFBPs with molecular weights 24,000 Mr, 28,000 Mr, 34,000 Mr or 43,000 Mr, whereas the 49,000 Mr IGFBP was more abundant in ER negative tumors (P less than 0.05). The IGFBP content was significantly (P less than 0.05) higher in five tumors than in their adjacent normal tissues suggesting that increased content of IGFBPs is a feature typically associated with the malignant transformation of breast tissue.

Human extrauterine decidual cells were studied for the presence of insulin-like growth factor-binding protein-1 (IGFBP-1) by using an avidin-biotin complex immunoperoxidase method with a purified monoclonal antibody 6303 against IGFBP-1. It is well established that human decidua expresses mRNA for IGFBP-1 and synthesizes and secretes this protein. We now describe, for the first time, that cells morphologically similar to uterine decidual cells found beneath the peritoneal mesothelium in inguinal hernia and in the cervical mucosa during normal pregnancy and in the mesenchymal tissue of the Fallopian tubes and ovaries at term react with the monoclonal antibody for IGFBP-1 equally to endometrial decidual cells. These results imply that the extrauterine mesenchymal cells, which have undergone decidual transformation, are not only morphologically but also functionally, similar to their counterparts in the uterus. Secondly, the data suggest that IGFBP-1 expression is associated with a special type(s) of cells and a certain stage of differentiation rather than special organs in the body.

Growing interest in IGF binding proteins and their roles in the modulation of IGF actions has prompted several laboratories to establish immunological assays to measure these proteins. Three distinct IGF binding proteins, referred as IGFBP-1, IGFBP-2 and IGFBP-3, have so far had their cDNAs cloned and sequenced. The availability of purified proteins and the development of polyclonal and monoclonal antibodies now allow specific quantitation of different IGFBP classes. This brief review will address on the current progress in the immunological assays of IGFBPs. The importance of the sensitivity, the reproducibility, and the standardization of the assays is discussed. Some additional information, like levels in amniotic fluid and serum of healthy adults and children, is given.