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Hypermutable Pseudomonas aeruginosa isolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutable P. aeruginosa isolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59  P. aeruginosa clinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterized P. aeruginosa strains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations in mutL or, less commonly, in mutS Hypermutable isolates also contained a range of mutations that are likely associated with adaptation of P. aeruginosa to the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutable P. aeruginosa strains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance., (Copyright © 2019 American Society for Microbiology.)

Background: Heavy menstrual bleeding (HMB) is an important cause of ill health in women and it accounts for 12% of all gynaecology referrals in the UK. Heavy menstrual bleeding is clinically defined as greater than or equal to 80 mL of blood loss per menstrual cycle. However, women may complain of excessive bleeding when their blood loss is less than 80 mL. Hysterectomy is often used to treat women with this complaint but medical therapy may be a successful alternative.The intrauterine device was originally developed as a contraceptive but the addition of progestogens to these devices resulted in a large reduction in menstrual blood loss. Case studies of two types of progesterone or progestogen-releasing systems, Progestasert and Mirena, reported reductions of up to 90% and improvements in dysmenorrhoea (pain or cramps during menstruation). Insertion, however, may be regarded as invasive by some women, which affects its acceptability as a treatment. Frequent intermenstrual bleeding and spotting is also likely during the first few months after commencing treatment., Objectives: To determine the effectiveness, acceptability and safety of progesterone or progestogen-releasing intrauterine devices in achieving a reduction in heavy menstrual bleeding., Search Methods: All randomised controlled trials of progesterone or progestogen-releasing intrauterine devices for the treatment of heavy menstrual bleeding were obtained by electronic searches of The Cochrane Library, the specialised register of MDSG, MEDLINE (1966 to January 2015), EMBASE (1980 to January 2015), CINAHL (inception to December 2014) and PsycINFO (inception to January 2015). Additional searches were undertaken for grey literature and for unpublished trials in trial registers. Companies producing progestogen-releasing intrauterine devices and experts in the field were contacted for information on published and unpublished trials., Selection Criteria: Randomised controlled trials in women of reproductive age treated with progesterone or progestogen-releasing intrauterine devices versus no treatment, placebo, or other medical or surgical therapy for heavy menstrual bleeding within primary care, family planning or specialist clinic settings were eligible for inclusion. Women with postmenopausal bleeding, intermenstrual or irregular bleeding, or pathological causes of heavy menstrual bleeding were excluded., Data Collection and Analysis: Potential trials were independently assessed by at least two review authors. The review authors extracted the data independently and data were pooled where appropriate. Risk ratios (RRs) were estimated from the data for dichotomous outcomes and mean differences (MD) for continuous outcomes. The primary outcomes were reduction in menstrual blood loss and satisfaction; in addition, rate of adverse effects, changes in quality of life, failure of treatment and withdrawal from treatment were also assessed., Main Results: We included 21 RCTs (2082 women). The included trials mostly assessed the levonorgestrel-releasing intrauterine device (LNG IUS) (no conclusions could be reached from one small study assessing Progestasert which was discontinued in 2001) and so conclusions are based only on LNG IUS. Comparisons were made with placebo, oral medical treatment, endometrial destruction techniques and hysterectomy. Ratings for the overall quality of the evidence for each comparison ranged from very low to high. Limitations in the evidence included inadequate reporting of study methods and inconsistency.Seven studies compared the LNG IUS with oral medical therapy: either norethisterone acetate (NET) administered over most of the menstrual cycle, medroxyprogesterone acetate (MPA) (administered for 10 days), the oral contraceptive pill, mefenamic acid or usual medical treatment where participants could choose the oral treatment that was most suitable. The LNG IUS was more effective at reducing HMB as measured by the alkaline haematin method (MD 66.91 mL, 95% CI 42.61 to 91.20; two studies, 170 women; I(2) = 81%, low quality evidence) or by Pictorial Bleeding Assessment Chart (PBAC) scores (MD 55.05, 95% CI 27.83 to 82.28; three studies, 335 women; I(2) = 79%, low quality evidence), improving quality of life and a greater number of women continued with their treatment at two years when compared with oral treatment. Although substantial heterogeneity was identified for the bleeding outcomes, the direction of effect consistently favoured the LNG IUS. There was insufficient evidence to reach conclusions on satisfaction. Minor adverse effects (such as pelvic pain, breast tenderness and ovarian cysts) were more common with the LNG IUS.Ten studies compared the LNG IUS with endometrial destruction techniques: three with transcervical resection, one with rollerball ablation and six with thermal balloon ablation. Evidence was inconsistent and very low quality with respect to reduction in bleeding outcomes and satisfaction was comparable between treatments (low and moderate quality evidence). Improvements in quality of life were experienced with both types of treatment. Minor adverse events were more common with the LNG IUS overall, but it appeared more cost effective compared to thermal ablation within a two-year time frame in one study.Three studies compared the LNG IUS with hysterectomy. The LNG IUS was not as successful at reducing HMB as hysterectomy (high quality evidence). The women in these studies reported improved quality of life, regardless of treatment. In spite of the high rate of surgical treatment in those having LNG IUS within 10 years, the LNG IUS was more cost effective than hysterectomy., Authors' Conclusions: The levonorgestrel-releasing intrauterine device (LNG IUS) is more effective than oral medication as a treatment for heavy menstrual bleeding (HMB). It is associated with a greater reduction in HMB, improved quality of life and appears to be more acceptable long term but is associated with more minor adverse effects than oral therapy.When compared to endometrial ablation, it is not clear whether the LNG IUS offers any benefits with regard to reduced HMB and satisfaction rates and quality of life measures were similar. Some minor adverse effects were more common with the LNG IUS but it appeared to be more cost effective than endometrial ablation techniques.The LNG IUS was less effective than hysterectomy in reducing HMB. Both treatments improved quality of life but the LNG IUS appeared more cost effective than hysterectomy for up to 10 years after treatment.

Purpose: The G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular., Experimental Design: In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome., Results: ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01)., Conclusions: In human tissues ADM expression is upregulated in cancer type-specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development., (©2013 AACR.)

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor-like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (Adm(AM+/Δ) animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both Adm(AM+/+) and Adm(AM+/Δ) mice. However, only Adm(AM+/Δ) animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in Adm(AM+/Δ) mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

Perinatal hemolytic disease occurs secondary to a hemolytic phenomenon of immune origin resulting in fetal or neonatal anemia. A 38-year-old pregnant woman was referred to the Department of high risk Obstetrics, Hospital Universitario La Paz Madrid because of presenting a dichorionic diamniotic twin pregnancy spontaneously, pre-pregnancy diabetes poorly controlled and severe alloinmunization anti-D. Her first pregnancy ended in a normal delivery at term; in the period of 4 years, she has three newborn with 36, 34 and 40 weeks respectively, who die with a week of life. After that, two intrauterine fetal death occur at 26 weeks of gestation. The patient who is RhD negative, suffers anti-D inmunization with a antibody titration of 1/1024 with 14 weeks of gestation. Twelve plasmapheresis, eight doses of anti-D inmunoglobulins and intrauterine transfusions has been the treatment received. A severe anemia is found during the ultrasound control of the middLe cerebral artery peak systolic velocity in both twins since the 16th week. It remains stable thanks to the treatment. Finally at the 28th week of gestation, pregnancy is terminated with a cesarean section. The twins are born alive and premature, but with good general state. The measurement of the middle cerebral artery peak systolic velocity predicts moderate-severe fetal anemia cases, which are the most important in the clinical management because of the need of active treatment or finish the pregnancy.

Adrenomedullin (AM) and its receptor subunit, calcitonin receptor-like receptor (CLR) are known to be important for endothelial function. The genotypes and phenotypes of AM and CLR in the endometrium were studied in relation to unexplained infertility. Endometrial biopsies from 12 fertile and 11 infertile women and blood samples from 156 fertile and 106 infertile women were collected. Protein and mRNA expression of AM and CLR was determined using immunohistochemistry and real time PCR. Allele and genotype frequencies in the AM (rs4399321 and rs7944706) and CLR genes (rs696574, rs1528233 and rs3771073) were performed using Taqman genotyping assays. Unexplained infertility was characterised by lower number of vessels stained with CLR in endometrium compared to fertile controls. There was no difference in AM expression. This could not be explained by SNP analysis in the AM or CLR genes. Imbalance in the AM/CLR system might alter endothelial function in women with unexplained infertility.

The human endometrium undergoes a unique process of benign angiogenesis under the control of ovarian steroids during reproductive life. Aberrant angiogenesis has been implicated in idiopathic menorrhagia, a common gynaecological complaint. One of the key factors involved in endometrial angiogenesis is adrenomedullin (AM), a multifunctional 52-amino acid peptide. AM mediates the activities of endometrial angiogenesis via calcitonin receptor-like receptor (CLR). The objective of the present study was to compare the endometrial expression of AM and CRL in women with and without idiopathic menorrhagia. Endometrial biopsies were obtained from 9 women with menorrhagia (≥80 ml per menstruation) and 12 women with normal blood loss (<80 ml per menstruation). Protein and mRNA expression levels of AM and CLR were determined using immunohistochemistry and real-time PCR. Compared to the controls, patients with menorrhagia exhibited low immunostaining intensity of AM, while high CLR staining was observed in the epithelium (p<0.05). No difference in mRNA expression was observed between the groups. These data suggest that an imbalance in the AM/CLR system might alter endometrial angiogenesis in menorrhagia.

The British Menopause Society Council is committed to provide up-to-date authoritative reviews to aid health professionals to inform and advise women about key issues in postreproductive health. This guidance refers to non-estrogen-based treatments for menopausal symptoms, such as hot flushes, symptoms of urogenital atrophy and lack of sexual desire. Treatment of choice should be based on up to-date information and targeted to individual women's needs. Non-hormonal strategies may be useful for women with estrogen-dependent disease such as breast cancer.

There has been some confusion among women and health professionals since the publication of the Women's Health Initiative and Million Women studies about the management of premature ovarian failure (POF). Both studies were undertaken in women aged 50 and over, and cannot be extrapolated to their younger counterparts, who would normally be producing their endogenous estrogen, since they have functioning ovaries. Estrogen-based replacement therapy is the main stay of treatment for women with POF and is recommended at least until the average age of natural menopause (52 years in the UK). This view is endorsed by regulatory bodies such as the Committee on Safety of Medicines (now the Commission on Human Medicines) in the UK. No evidence shows that estrogen replacement increases the risk of breast cancer to a level greater than that found in normally menstruating women, and women with POF do not need to start mammographic screening early unless other risk factors are present, such as family history.

Purpose: The role for the hypoxia-inducible angiogenic factor adrenomedullin (AM) in tumor growth and progression has been suggested. Calcitonin receptor-like receptor (CL) is a G protein-coupled receptor (GPCR) that mediates effects of AM, but little information is available on its expression and functional state in human tumors. The present study attempted to determine CL potential for antiangiogenic therapy of uterine leiomyoma., Experimental Design and Results: GPCR CL is transported to the cell surface and recognized by AM only when terminally/mature glycosylated. The presence and localization of this form of the receptor in tumor and surrounding myometrial tissues obtained from leiomyoma-bearing uteri were examined using deglycosylation, immunoblotting, and immunofluorescence analysis. The mature CL glycoprotein was expressed in both tissues and localized exclusively in normal and tumor endothelium within leiomyoma-bearing uteri. The functionality of the receptor expressed in myometrial microvascular endothelial cells (MMVEC) was examined in vitro using receptor internalization and angiogenic assays. The mature CL glycoprotein expressed by primary MMVECs was functional because AM interacted with this GPCR and induced its internalization as well as angiogenic effects (proliferation and migration) in MMVECs in vitro. Finally, the levels of tissue-expressed mature CL glycoprotein as a functional form of this GPCR were analyzed by immunoblotting. The expression of this functional form of the receptor in vivo was significantly decreased (P = 0.01) in leiomyoma tissue, and this was concurrent with the decrease in microvascular density (measured by Chalkley counting) in tumor compared with surrounding myometrium (P = 0.031)., Conclusions: Our findings suggest that GPCR CL mediates angiogenic effects of AM in myometrium and that further evaluation of the properties of the CL expressed in both normal and tumor endothelium in vivo may be essential before targeting this endothelial GPCR for antiangiogenic therapies.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are related peptides with distinct pharmacological profiles. Calcitonin-receptor-like receptor (CRLR, now known as CL) can function as either an AM receptor or a CGRP receptor, when cotransfected with receptor-activity-modifying proteins (RAMPs) that define ligand-binding specificity. The aim of the present study was to determine the role of endogenously expressed CL (EndoCL) in generating endogenous AM and CGRP receptors. We raised anti-human CL antibody and identified microvascular endothelial cells (MVECs) as a major CL-expressing cell type in tissues by immunohistochemistry. Cultured MVECs continue to express EndoCL as well as fully active endogenous AM- and CGRP-sensitive receptors in vitro, as demonstrated by the ability of both peptides to induce migration and Akt phosphorylation. We therefore tested the hypothesis that endothelial EndoCL can interact with both AM and CGRP by examining receptor internalisation and desensitisation (loss of the ability to induce Akt phosphorylation). We found that agonist-mediated internalisation of EndoCL occurs in response to AM but not CGRP in MVECs. However, AM-induced EndoCL internalisation was blocked by antagonists of both AM and CGRP receptors: AM(22-52) and CGRP(8-37), respectively. Furthermore, AM-induced EndoCL internalisation resulted in desensitisation not only of AM but also of CGRP receptors. Finally, CGRP also induced desensitisation of both endogenous AM and CGRP receptors, but did not mediate EndoCL internalisation despite interaction with this receptor. Thus, EndoCL interacts with both AM and CGRP, and simultaneously acts as a receptor for both peptides (i.e acting as an endogenous AM/CGRP receptor) in endothelial cells. Interaction with either ligand is sufficient to induce EndoCL desensitisation to both AM and CGRP, but differential mechanisms are involved since only AM induces EndoCL internalisation. These novel findings regarding regulation of EndoCL function in endothelial cells are likely to be of importance in conditions where AM or CGRP levels are elevated, such as cardiovascular disease, diabetes and inflammation.

The angiogenic activity of peptide adrenomedullin (AM) was first shown in 1998 . Since then, a number of reports have confirmed the ability of AM to induce the growth and migration of isolated vascular endothelial and smooth muscle cells in vitro and to promote angiogenesis in xenografted tumours in vivo. In addition, knockout murine models point to an essential role for AM in embryonic vasculogenesis and ischaemic revascularisation. AM expression is upregulated by hypoxia (a typical feature of solid tumours) and a potential role as a regulator of carcinogenesis and tumour progression has been proposed based on studies in vitro and in animal models. Nevertheless, translational research on AM, and in particular, confirmation of its importance in the vascularisation of human tumours has lagged behind. In this commentary, we review current progress and potential directions for future research into the role of AM in tumour angiogenesis.

The British Menopause Society Council aims to help health professionals inform and advise women about the menopause. This guidance regarding estrogen-based hormone replacement therapy (HRT), including tibolone, which is classified in the British National Formulary as HRT, responds to the results and analysis of the randomized Women's Health Initiative studies and the observational Million Women Study. Treatment choice should be based on up-to-date information and targeted to individual women's needs. HRT still offers the potential for benefit to outweigh harm, providing the appropriate regimen has been instigated in terms of dose, route and combination.

Background: This study was designed to assess the long-term efficacy (5 years) of the levonorgestrel-releasing intrauterine system (LNG-IUS) in protecting the endometrium from hyperplasia during estrogen replacement therapy in perimenopausal women., Methods: Prospective, open, outpatient clinical trial in London and Oxford. Eighty-two women received oral conjugated equine estrogen 1.25 mg daily and LNG-IUS releasing 20 mug levonorgestrel per 24 h. Endometrial biopsy and histological assessment were performed annually. Endometrial thickness was measured by vaginal ultrasonography., Results: Non-proliferative endometrium was present at the end of cycles 12, 24, 36, 48 and 60 in 98.6, 98.6, 95.5, 96.8 and 95.2% of the participants respectively. No endometrial hyperplasias were confirmed throughout a period of 60 cycles. The proportion of amenorrhoeic women increased from 54.4% at 12 cycles to 92.7% at the end of the study. The continuation rate per 100 women at 60 cycles was 79.84 (95% CI 71.0-88.6)., Conclusions: The LNG-IUS with estrogen supplementation in perimenopausal women suppresses endometrial proliferation resulting in amenorrhoea and relieves vasomotor symptoms. The treatment regimen is well tolerated and provides an alternative strategy for perimenopausal women with the likelihood of increasing compliance.

The British Menopause Society Council aims to help health professionals to inform and advise women about the menopause. The oestrogen plus progestogen arm of the Women's Health Initiative was stopped in July 2002. This guidance regarding hormone replacement therapy (HRT) use responds to the results and analysis that have been published since then, as well as the Million Women Study published in August 2003. Because there are few effective alternatives to HRT for vasomotor and urogenital symptoms, oestrogen-based treatments still have a major role. HRT is also most effective for the prevention of osteoporosis. Unopposed oestrogens are contraindicated in women with an intact uterus, and hence a range of oestrogen and progestogen combinations, with differing routes of delivery, now exists under the title of "HRT". Treatment choice should be based on up to date information and targeted to individual women's needs. Hormone replacement still offers the potential for benefit to outweigh harm, providing the appropriate regimen has been instigated in terms of dose, route and combination.

Objective: Two randomized comparative multicenter studies were conducted to establish the endometrial safety and tolerability of a triphasic sequential hormone replacement estradiol valerate/medroxyprogesterone acetate (E2V/MPA) therapy regimen., Methods: Study 1 was a randomized, double-blind, clinical phase III study in 399 postmenopausal women, following parallel-group design with two groups. The duration of study treatment was 12 or 13 cycles of 28 days. A double-dummy technique was used to ensure blinding in the study. The investigational drugs were E2V/MPA triphasic and E2V/MPA biphasic (Diviseq and Divina, respectively; Orion Pharma). In study 2, a total of 341 subjects were randomly allocated by computer into two parallel groups receiving either E2V/MPA or estradiol/norethisterone acetate triphasic (E2/NETA, Trisequens; Novo Nordisk A/S) for 12-13 cycles. The study was an open, clinical phase III trial with a randomized, parallel-group design. Endometrial biopsies combined with transvaginal ultrasound were undertaken before and at the end of treatment during the progestogen phase. Bleeding patterns and symptom control were assessed throughout both studies., Results: E2V/MPA triphasic was found to have similar endometrial effects and bleeding patterns to those with E2V/MPA biphasic and E2/NETA triphasic. Climacteric symptoms were relieved as quickly and effectively as with the two comparator treatments. No adverse drug reactions specific to E2V/MPA triphasic were observed. At the end of the study, the proportions of secretory samples were 67.1% for the combined E2V/MPA triphasic groups, 65.6% for the E2V/MPA biphasic group and 71.6% for the E2/NETA triphasic group. One case of hyperplasia occurred in the E2V/MPA triphasic group. Thus the incidence of hyperplasia for the combined groups was 0.33%., Conclusions: The triphasic E2V/MPA regimen was well tolerated and produced endometrial effects similar to those of the two comparators. Extending estrogen during the so-called treatment-free week with a lower dose of estradiol was effective in controlling vasomotor symptoms.

The growing influence of the mass media on public understanding of health care matters has increased both information and misinformation in patients seeking help with menopausal symptoms. The use of shared decision-making strategies provides the opportunity to engage the patient in taking some responsibility for their own treatment. It also allows the identification and correction of any distortions in the perceived balance of evidence for and against any particular treatment. Shared decision-making balances the need to respect patients' values and autonomy with the drive towards evidence-based medicine and clinical cost-effectiveness. Although ways of achieving such a balance are much discussed, the current need is for research which can identify effective strategies that allow the principles of "shared decision-making" and "evidence-based patient choice" to be validated and applied in clinical practice. Previous research focused on hormone replacement therapy indicates that the patient's decision is the outcome of the way they balance the pros and cons of taking (or not taking) it, and that their satisfaction with the decision is strongly associated with the perception that they have been given information about the full range of treatment available. It therefore seems likely that patients will respond more positively to consultations which include discussion of hormone replacement therapy alongside alternative strategies such as natural remedies and dietary/lifestyle changes. Psychological factors influencing treatment choice and the decision making process are discussed, and clinical and research implications for clinical practice in the menopause clinic examined.

Adrenomedullin is a 52 amino acid peptide originally isolated from human phaeochromocytoma in 1993. It was initially demonstrated to have profound effects on the vasculature including vasodilatation and subsequently promotion of angiogenesis. Since then it has become apparent that it has a wide range of other biological actions including regulation of cell growth and differentiation. Successful pregnancy outcome relies on establishing and maintaining throughout gestation an efficient blood supply to the fetus. This allows the exchange of nutrients, oxygenation of fetal blood and removal of cytotoxins from the fetus, such as carbon dioxide. One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. Evidence now points towards a vital role for adrenomedullin in the regulation of placentation. It appears that adrenomedullin may play important roles in the regulation of fetal perfusion both in normal and in compromised pregnancies. However, most studies have focused on measuring adrenomedullin levels and studying its expression as well as that of its receptors. More functional studies are now required to elucidate the underlying mechanisms involved.

Adrenomedullin (ADM) is an angiogenic factor that has also been shown to be a mitogen and a hypoxia survival factor for tumour cells. These properties point to ADM as a potential promoter of human malignancies, but little data are available concerning the expression of ADM in human breast cancer. In the present work, we have examined ADM peptide expression in a series of malignant breast tumours by immunohistochemistry using a newly developed anti-ADM monoclonal antibody. In addition, ADM plasma concentrations in breast cancer patients and healthy controls were determined by radioimmunoassay. Of the examined breast cancer samples, 27/33 (82%) showed a moderate to strong staining intensity. ADM-peptide expression in breast tumours was significantly correlated with axillary lymph node metastasis (P=0.030). Analysis of ADM plasma concentrations showed no significant difference between the circulating ADM levels of breast cancer patients and healthy controls. However, a significant positive correlation was found between tumour size and plasma ADM levels (r=0.641, P=0.017). Moreover, ADM levels in breast cancer patients correlated with the presence of lymph node metastasis (P=0.002). In conclusion, we have shown for the first time that ADM peptide is widely expressed in breast cancer and that the degree of expression is associated with lymph node metastasis. ADM peptide in plasma of breast cancer patients reflects the size of the primary tumour, but is unlikely to be a useful tumour marker for the detection of breast cancer. Plasma ADM might represent an independent predictor of lymph node metastasis. The clinical implications of these findings remain to be evaluated.

The British Menopause Society Council aims to aid health professionals to inform and advise women about the menopause. The oestrogen plus progestogen arm of the Women's Health Initiative was stopped in July 2002. This guidance regarding hormone replacement therapy (HRT) use responds to the results and analysis that have been published since then. Because there are few effective alternatives to HRT for vasomotor and urogenital symptoms, oestrogen-based treatments still have a major role. HRT is also most effective for prevention of osteoporosis. Unopposed oestrogens are contraindicated in women with an intact uterus, and hence a range of oestrogen and progestogen combinations, with differing routes of delivery, now exists under the title of "HRT". Treatment choice should be based on up to date information and targeted to individual women's needs. Hormone replacement still offers the potential for benefit to outweigh harm, providing the appropriate regimen has been instigated in terms of dose, route and combination.

Peri and postmenopausal bleeding, with or without the use of hormone replacement therapy, is a common clinical problem. The exclusion of endometrial hyperplasia and carcinoma is the key issue in the evaluation of patients with abnormal uterine bleeding. Transvaginal ultrasound measurement of endometrial thickness has become a routine procedure and an initial investigation in patients with abnormal uterine bleeding. There is debate as to whether a cut-off of 5 or 4 mm endometrial thickness should be employed. If the endometrial thickness is above these values, polyps have been diagnosed or the patient is presenting with recurrent bleeding, endometrial disease has to be excluded by histological assessment. Outpatient aspiration curettage has superseded dilatation and curettage, which was previously considered to be the gold standard for obtaining endometrial tissue, and provides the same sensitivity in detecting endometrial disease. Hysteroscopy allows visualisation of the uterine cavity and the opportunity for targeted biopsy and removal of endometrial polyps.

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.

Menorrhagia is defined as a 'complaint of heavy cyclical menstrual bleeding occurring over several consecutive cycles'. Objectively it is a total menstrual blood loss equal to or greater than 80 ml per menstruation. It is estimated that approximately 30% of women complain of menorrhagia. Excessive bleeding is the main presenting complaint in women referred to gynecologists and it accounts for two-thirds of all hysterectomies, and most of endoscopic endometrial destructive surgery. Thus, menorrhagia is an important healthcare problem. Its etiology, investigation, medical and surgical management are described. In approximately 50% of cases of menorrhagia no pathology is found at hysterectomy. Abnormal levels of prostaglandins or the fibrinolytic system in the endometrium have been implicated. Effective medical treatments suitable for long-term use include intrauterine progestogens, antifibrinolytic agents (tranexamic acid) and nonsteroidal anti-inflammatory agents (mefenamic acid). Over the past decade there has been increasing use of endometrial destructive techniques as an alternative to hysterectomy. Their further refinement and the advent of fibroid embolization has increased the options available to women.

Progestogens are added to oestrogen in hormone replacement therapy regimens to reduce the risk of endometrial cancer. We have performed in vitro studies analysing gene expression of isolated normal endometrial epithelia cells (NEE) treated with estradiol and the progestogen norethisterone acetate (NETA). We report here for the first time upregulation of the Wnt-7a gene by NETA in estrogen treated NEE. Wnt genes are a large family of developmental genes associated with cellular responses such as oncogenesis. We therefore suggest that upregulation of Wnt-7a may be associated with the antineoplastic effects of progestogens on the endometrium.

The angiogenic peptide adrenomedullin (ADM) has been implicated as a mediator of the increased risk of endometrial hyperplasia and cancer resulting from the use of tamoxifen for the treatment and prevention of breast cancer. ADM has been shown to be induced by tamoxifen in the endometrium and to be a growth factor for endometrial endothelial cells in vitro. We have now shown ADM to be strongly angiogenic in the mouse subcutaneous sponge angiogenesis assay. To examine the role of ADM in tumor growth, the ADM cDNA was transfected into endometrial carcinoma cells followed by xenografting into athymic mice. Two endometrial cancer cell lines were employed, those in which transfection and expression of ADM resulted in no effect on growthin vitro (Ishikawa cells) and those in which expressionof exogenous ADM stimulated in vitro growth (RL95.2 cells). A clear enhancement of tumor growth was seen with both cell lines but the effect was far greater with the RL95.2 cells. We conclude that ADM is pro-tumorigenic by stimulating either angiogenesis alone or by stimulating angiogenesis and carcinoma cell growth directly. The combined activities lead to a striking increase in tumor growth. These results provide the first direct evidence of tumorigenic activity of ADM and provide further support for ADMs involvement in tamoxifen induced endometrial neoplasia.

Tamoxifen is the current therapy of choice for patients with oestrogen receptor positive breast cancer, and it is currently under evaluation as a prophylactic for women at high risk of developing the disease. However, tamoxifen is also known to induce proliferative changes in the endometrium increasing the risk of developing endometrial hyperplasia, polyps and carcinoma. Angiogenesis is an intimate part of this process. For this reason, we have examined the expression of several well characterized angiogenic factors, namely, acidic and basic fibroblast growth factor, thymidine phosphorylase, vascular endothelial growth factor and adrenomedullin in both normal and tamoxifen exposed pre- and postmenopausal endometrium. Vascular density and endothelial proliferation index were also quantified. We found increased expression of acidic and basic fibroblast growth factor and adrenomedullin after treatment with tamoxifen mainly in premenopausal tissue. Vascular density was significantly increased in pre- but not post-menopausal endometrium (P=0.0018) following tamoxifen treatment. These results support the notion that angiogenesis is integral to the response to tamoxifen exposure, and is a potential target with which to block these side effects of tamoxifen., (Copyright 2002 Cancer Research UK)

Background: Progestogens are used clinically for contraception, to control excessive menstrual bleeding, and to prevent estrogen-induced endometrial hyperplasia. A significant problem with progestogen-only methods of contraception is the induction of breakthrough bleeding., Methods: The effects of different progestogens on angiogenesis were examined using two approaches. The mouse sponge angiogenesis assay employed direct delivery of the dose ranges achieved therapeutically. The angiogenic response to long-term intrauterine levonorgestrel exposure, compared with unexposed premenopausal endometrium, was also studied., Results: In the mouse sponge assay, norethisterone and medroxyprogesterone acetate stimulated angiogenesis at all doses, but was dose-dependent for levonorgestrel and nomegestrol. Levonorgestrel stimulated angiogenesis in the dose range 100 pmol/l to 10 nmol/l, but not at higher doses. In contrast, nomegestrol acetate stimulated angiogenesis at high, but not low, doses. Expression of acidic and basic fibroblast growth factors, thymidine phosphorylase, vascular endothelial growth factor and adrenomedullin were unaltered in levonorgestrel-exposed endometrium compared with premenopausal controls. Vascular density was increased but endothelial proliferation reduced in levonorgestrel-exposed endometrium., Conclusions: This is the first report of the direct effects of a wide range of doses of different progestogens on angiogenesis; results suggest that vascular targeting may be an effective strategy to deal with progestogen-induced abnormal bleeding.

Progestogens are used clinically for contraception, to control excessive menstrual bleeding and to oppose oestrogen in hormone replacement therapy. The use of intrauterine levonorgestrel (LNG) is however, associated with endometrial atrophy and decidualisation of the stroma. In this study, we aimed to identify genes whose expression is modulated by LNG either alone or in combination with progesterone. Thus endometrial stromal cells were stimulated with progesterone, LNG or LNG and progesterone. Poly-A RNA was isolated and used to probe expression arrays. The expression of a number of genes was altered on exposure to LNG or LNG and progesterone. Alteration of expression patterns was confirmed using semi-quantitative RT-PCR and western blot analysis. In particular, the protease activated receptor-1 (PAR-1) gene that encodes a receptor for thrombin was down regulated. This is the first demonstration that PAR-1 is down regulated by the progestogen LNG in human endometrium. Alteration in the expression levels of this receptor may affect both growth and haemostatic activity within the endometrium and may account for the observed morphological effects seen in users of intrauterine LNG delivery devices.

The calcitonin receptor-like receptor (CRLR) can function as a receptor for either calcitonin gene-related peptide (CGRP) or adrenomedullin (AM), depending upon co-expression with members of a novel family of receptor activity-modifying proteins (RAMP). RAMP1 presents the CRLR at the cell surface as a CGRP/AM receptor. RAMP2- and RAMP3-transported CRLR receptors act as AM-specific receptors. However, it is still unknown if this signalling system operates in vivo. Of particular interest is the uterus, where both peptides and their binding sites are known to be present and where both mitogenic and vasodilatory responses to AM and CGRP have been demonstrated. In this study, we examined whether CRLR and RAMP are co-expressed in the same populations of cells in human uterine tissue. Analysis by in-situ hybridization and immunocytochemistry revealed a heterogeneous and cell type-specific distribution of components of this AM/CGRP signalling system. Adrenomedullin mRNA was expressed and evenly distributed across all cell types. CRLR mRNA was predominantly found in blood vessels. RAMP1 expression was specific to myometrial myocytes and vascular smooth muscle cells in uterine arteries. RAMP2 and RAMP3 mRNA were not detectable by in-situ hybridization. The pattern of differential and cell-specific expression of CRLR and RAMP suggests the involvement of CRLR/RAMP1 in the processes of vasodilation, smooth muscle relaxation and angiogenesis in response to AM and CGRP in the human uterus, but also indicates that other receptors may be implicated.

Regions of hypoxia are a common feature of solid tumours. When tumour cells are exposed to hypoxic stress, transcription of a battery of genes is initiated. The angiogenic factor adrenomedullin (ADM) is a hypoxia regulated gene. ADM is thought to act through the G protein-coupled receptor calcitonin receptor-like receptor (CRLR), with specificity being conferred by the receptor associated modifying protein 2 (RAMP2). Here we report for the first time that ADM treated or stably transfected Ishikawa cells overexpressing ADM show increased resistance to hypoxia induced apoptosis. These cells also show an upregulation of the oncoprotein Bcl-2, which is protective against hypoxic cell death when transiently transfected into Ishikawa cells. Since Ishikawa cells express the putative ADM-receptor CRLR-RAMP2 the production and secretion of ADM with the consecutive upregulation of Bcl-2 could establish an autocrine/paracrine mechanism rescuing malignant cells from hypoxic cell death. These results, taken together with our previous findings that ADM is an angiogenic factor which is upregulated by the nonsteroidal antiestrogen tamoxifen (TAM) in endometrial cells, implicate this peptide as a promoter of tumour growth and a possible target for anticancer strategies.

Human endometrium is a mucosa served by a microvascular blood supply that involves benign angiogenesis under the control of ovarian steroids throughout reproductive life. Adrenomedullin is a multifunctional 52-amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, vasodilation and smooth muscle relaxation. We have previously shown that adrenomedullin is present in the human uterus. To investigate further the role of adrenomedullin in human endometrial angiogenesis, a method for the isolation and culture of non-pregnant endometrial endothelium was developed. Enzymatic dispersion and 'Percoll' gradient centrifugation, followed by positive selection using Ulex europaeus agglutinin-coated immunomagnetic beads, yielded pure isolates of endothelium. The cells formed a typical 'cobblestone' monolayer within 5-7 days and expressed the classic endothelial markers, CD31 and von Willebrand factor. The presence of adrenomedullin immunoreactivity in endometrial endothelial cells was shown by immunohistochemistry both in vitro and in vivo. Adrenomedullin promotes growth of endothelial cells as measured by [methyl-(3)H] thymidine uptake. Adrenomedullin also induced cyclic AMP in endometrial endothelial cells. These results demonstrate, for the first time, that adrenomedullin is an autocrine growth factor for human endometrial endothelial cells and is thus involved in endometrial angiogenesis.

Uterine leiomyomas are the most prevalent benign tumor type in women of reproductive age and are one of the most common indications for hysterectomy. The expression of five angiogenic factors, adrenomedullin (ADM), vascular endothelial growth factor (VEGF), acidic fibroblast growth factor, basic fibroblast growth factor, and platelet-derived endothelial cell growth factor/thymidine phosphorylase, were examined in 91 uteri collected throughout the menstrual cycle; 52 of which contained leiomyomata, and the remainder were normal controls. The microvascular density and endothelial proliferative indices were then determined for each of the uterine sections. ADM and VEGF were the most widely expressed angiogenic factors in the leiomyomas. Furthermore, the expression of ADM and VEGF in the endometrium and myometrium was up-regulated in leiomyoma-bearing uteri compared with controls. Although acidic fibroblast growth factor and basic fibroblast growth factor were expressed in leiomyomas and endometrium in all of the uterine samples examined, they were only expressed in the myometrium of leiomyomata-bearing uteri. Endothelial proliferation in leiomyomas was statistically greater than that of the myometrium and endometrium, both within and between uteri (P < 0.05). The vascular density in the myometrium but not the endometrium was significantly increased in leiomyoma-containing uteri (P < 0.05). Expression of ADM alone correlated directly with vascular density and endothelial cell proliferation index in leiomyomas and myometrium and may account for the high vascularity found in leiomyomas and the myometrium of leiomyoma-bearing uteri. As such, ADM is identified as a novel target for antiangiogenic therapy of these benign, clinically problematic uterine tumors.

Steroids are commonly employed in current clinical practice. The benefits of steroids in hormone replacement therapy, contraception and prevention or treatment of breast cancer are limited by their side effects arising from disorders in endometrial function. These side effects are complex and enclose bleeding problems and endometrial proliferation during hormone replacement therapy and antioestrogen treatment or menstrual disturbances during oral contraception. Numerous reports have identified gene targets influenced by steroids and have implicated these products as contributors to endometrial physiology or pathology. The expression of estrogen and progesterone receptors is regulated by steroids. The new estrogen receptor (ER) subtype ERbeta with different functional characteristics from ERalpha was recently described in endometrium. In addition, there is now increasing evidence that the functionally distinct progesterone receptor (PR) isoforms A and B are differentially expressed in this tissue. The relative proportions of these steroid receptors and their interaction determine the expression of specific genes upon steroidal stimulation. Steroids induce endometrial expression of various growth and angiogenic factors. Dysregulations of this steroid modulated expression is believed to be involved in the pathogenesis of many endometrial diseases. Irregular bleeding induced by steroidal contraception, for example, is thought to involve aberrant endometrial vascular development and expression of angiogenic growth factors. The antioestrogen tamoxifen induces growth factors like vascular endothelial growth factor and adrenomedullin which may be key mediators of endometrial neoplastic effects. This review describes recent advances regarding the mechanism of action of steroids on endometrium. The expression of oestrogen and progesterone receptors as well as steroid hormone dependent growth factors and angiogenic modulators are going to be discussed.

Background: Hormone replacement therapy (HRT) is commonly prescribed to treat menopausal symptoms and to prevent post-menopausal bone loss. However, many women are concerned about hormonal replacement therapy because they believe that such treatment will result in weight gain. The effect of HRT on weight and body fat distribution has not yet been examined in systematic reviews. It is an important topic since many women decline oestrogen therapy due to their concerns about resultant weight gain, and thus forego its potential therapeutic benefits., Objectives: To evaluate the effect of unopposed oestrogen or combined oestrogen and progestogen hormone replacement therapy (HRT) upon the weight and body fat distribution of perimenopausal and postmenopausal women., Search Strategy: The search strategy of the Menstrual Disorders and Subfertility Group was used for the identification of randomised controlled trials (RCTs). Computerised searches of MEDLINE, EMBASE, Current Contents, Biological Abstracts and CINAHL were performed. Attempts were made to identify trials from citation lists of review articles and relevant papers already obtained. In most cases, first authors of each eligible trial were contacted for additional information. All those trials that had been located as at August 1998 were examined for eligibility., Selection Criteria: All randomised, placebo or no treatment controlled trials that detailed the effect of HRT on weight or body fat distribution, including studies where HRT was combined with other therapy such as diet, supplements or exercise. Studies were eligible for consideration even though the main focus of the trial may have been on another aspect of HRT. Previous HRT use should have ceased at least one month (in the case of patches, cream or gel) or three months (for oral preparations or subcutaneous pellets) before commencement of the study., Data Collection and Analysis: Twenty two RCTs were identified that fulfilled the inclusion criteria for this review. The results of one trial were not available in a form that allowed it to be included in the meta-analysis; however, it has been included in the text of the review for discussion. Twenty four RCTs are awaiting assessment pending additional information from first authors. Two reviewers extracted the data independently, and the weighted mean differences for continuous outcomes were estimated from the data. Results for unopposed oestrogen and combined oestrogen were analysed separately, and the effect of each treatment regimen on body weight, BMI, waist-hip ratio, fat mass and skinfold measurement was examined where available. The effect of differing dosage levels on these parameters was also examined., Main Results: Outcomes were evaluated separately for unopposed oestrogen and oestrogen/progestogen regimens. Statistical analysis was performed using the weighted mean difference for continuous outcomes as recommended by the Cochrane Menstrual Disorders and Subfertility Group. No statistically significant difference was found in mean weight gain between those using unopposed oestrogen and non-HRT users (0.66 kg, 95% CI -0.62, 1.93). No significant difference was found in mean weight gain between those using oestrogen/progestogen therapy and non-HRT users (-0.47 kg, 95% CI -1.63, 0.69). Insufficient data exist to enable meta-analysis of the effect of unopposed oestrogen on BMI. The reviewers found no statistically significant difference in mean BMI increase between those using oestrogen/progestogen and non-HRT users (-0.50, 95% CI -1.06, 0.06). Insufficient data exist to enable meta-analysis of the effect of HRT on waist-hip ratio, fat mass or skinfold thickness., Reviewer's Conclusions: There is evidence of no effect of unopposed oestrogen or combined oestrogen on body weight, indicating that these regimens do not cause extra weight gain in addition to that normally gained at menopause. (ABSTRACT TRUNCATED)

Transient transfection of COS-1 cells followed by fixation, embedding in paraffin, and immunohistochemistry has identified anti-vascular endothelial growth factor (anti-VEGF) mouse monoclonal antibodies that efficiently immunostain VEGF in paraffin-embedded tissue sections. Immunohistochemical localization of VEGF in 34 specimens of normal human endometrium that had been collected at different stages of the menstrual cycle was then performed. VEGF was present at all stages of the cycle, but both the pattern and the intensity of staining varied. Thus, VEGF expression occurred predominantly in the endometrial epithelium and while weak in the proliferative phase, was strong in the secretory phase. VEGF expression in the stroma was weaker than in the proliferative phase glands and did not change throughout the cycle. These findings are in agreement with reports of VEGF mRNA expression in the endometrium, but disagree with previous immunohistochemical studies that employed an immunohistochemically unvalidated antiserum. This study has shown that the commercially available anti-VEGF monoclonal antibody M293 is excellent for the immunohistochemical localization of VEGF in paraffin sections.

Tamoxifen is currently the most widely used drug for the treatment of breast cancer, but there now exists considerable evidence that tamoxifen can also induce endometrial hyperplasia in pre menopausal women. We have used PCR differential display on primary human endometrial isolates in an attempt to identify genes induced by tamoxifen but not estrogen. Eight such differentially expressed bands were cloned and sequenced, one of which was found to be the peptide adrenomedullin. We have shown that adrenomedullin is a novel growth factor for endothelial cells and is angiogenic in vivo in the chick chorioallantoic membrane assay. Immunohistochemical analysis of endometrial sections have shown that while macrophages in the endometrium express adrenomedullin at a low level, endometrial macrophages of women receiving tamoxifen strongly express adrenomedullin (P=0.008). We postulate that endometrial induction of the angiogenic factor adrenomedullin by tamoxifen is part of the mechanism by which tamoxifen results in endometrial hyperplasia.

Human endometrial vasculature has the unique property of undergoing benign angiogenesis during each menstrual cycle under the influence of the ovarian steroids estradiol and progesterone. However, neither has intrinsic angiogenic activity and endometrial angiogenesis involves stimulation by ovarian steroids of angiogenic factor release by the epithelium and stroma which then act on the endothelium. In vitro models using cultures of isolated endometrial epithelium, stroma and endothelium now allow mechanistic questions to be addressed. Vascular endothelial growth factor and platelet-derived endothelial cell growth factor/thymidine phosphorylase at present appear to be key players in endometrial angiogenesis.

The angiogenic enzyme platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) was strongly expressed in the endometrial glands in the luteal and menstrual, but not the proliferative, phases of the cycle. The converse was seen in the stroma, where expression was strong in the proliferative, but not the luteal or menstrual, phases. Inflammatory cytokines induced PD-ECGF/TP expression in primary cultures of human normal endometrial epithelial (NEE) and normal endometrial stromal cells. The profile of cytokine induction of PD-ECGF/TP was cell dependent. Thus, in NEE cells, PD-ECGF/TP expression was strongly induced by the combination tumor necrosis factor-a and interferon-gamma. In contrast, in normal endometrial stromal cells, interferon-gamma gave, by far, the strongest induction of PD-ECGF/TP. Expression of the enzyme was not regulated by ovarian hormones alone. Although treatment of NEE cells with a physiological concentration of progesterone (5 X 10[-8] M) or transforming growth factor-beta1 (10 ng/ml) alone had no effect on PD-ECGF/TP expression, when delivered together at the same dose they induced a 48-fold increase in expression. This expression correlates with cyclic changes in progesterone and transforming growth factor-beta1 levels in the uterus.

Wnt genes are transforming to mouse breast epithelium and are hormonally regulated in vivo. To assess their role in another endocrine-responsive human cancer, the expression of seven Wnt genes (Wnt 2, 3, 4, 5a, 7a, 7b and 10b) in normal human endometrium and endometrial cells, and endometrial carcinoma tissues and cell lines was investigated by ribonuclease protection analysis. Wnt2, 3, 4 and 5a mRNAs but not Wnt7a, 7b or 10b mRNAs were expressed in primary culture of normal endometrial epithelial (NEE) and stromal (NES) cells. In contrast, in four endometrial carcinoma cell lines (RL95-2, HEC-1-A, AN3 CA and Ishikawa), Wnt2 and Wnt3 mRNAs were absent. Wnt4 was expressed in only one out of four cell lines (RL95-2), and Wnt5a was much lower. Wnt7a and Wnt7b mRNAs were expressed in three out of four cell lines (RL95-2, HEC-1-A and Ishikawa). Wnt10b mRNA was expressed in RL95-2 and AN3 CA. In fresh tissues, all Wnt genes apart from Wnt10b were expressed in normal endometrium and endometrial carcinoma. Similar to the cell lines, the level of Wnt4 mRNA expression was significantly higher in the normal endometrium than endometrial carcinoma. Wnt2, 3 and 5a mRNAs were also lower in endometrial carcinoma compared with normal endometrium. There was no difference in the level of Wnt2, 3, 4 and 5a mRNA expression between proliferative phase and secretory phase of the menstrual cycle, or between either menstrual phase and the first trimester of pregnancy. In vitro, progesterone and/or 17beta-oestradiol had no effect on Wnt2, 3, 4, 5a and 7b mRNA expression in NES and all endometrial carcinoma cell lines. The data indicate that all Wnt genes were expressed in vitro, six out of seven Wnt genes (Wnt 2, 3, 4, 5a, 7a and 7b) were expressed endogenously in the human endometrium, their mRNA expression was hormonally independent and Wnt4 gene down-regulation as well as down-regulation of Wnt 2, 3 and 5a may be associated with endometrial carcinoma.