Comparison of Bayer Advia Centaur immunoassay results obtained on samples collected in four different Becton Dickinson Vacutainer tubes.

BACKGROUND: Medicines and Healthcare products Regulatory Agency's (MHRA's) Medical Device Alert MDA/2004/048 described bias in some endocrine test results obtained on a few immunoassay platforms, particularly the Bayer Advia Centaur instrument, when using blood specimens collected into Becton Dickinson (BD) Vacutainer SSTII Advance tubes. As users of BD tubes and the Advia Centaur instrument, we addressed our concerns about the quality of the results that we had previously reported by undertaking an independent study. METHOD: We compared the results of 15 immunoassays performed on Bayer Advia Centaur using blood specimens collected into four different BD Vacutainer tubes (plain, old and newly released BD SSTII Advance, and BD PSTII). RESULTS: Compared with plain tubes, old SSTII Advance tube results showed no bias for testosterone, CA15-3, follicle-stimulating hormone and folate assays, but gave a positive bias for cortisol and a negative bias for vitamin-B12. Compared with plain tubes, BD PSTII tubes gave no significant bias for thyroid function tests, prolactin, parathyroid hormone, and CA125, but gave a negative bias for steroid assays, and a positive bias for gonadotrophins. The results obtained using new BD SSTII Advance tubes were generally comparable with those on plain tubes. CONCLUSIONS: Only for cortisol did our findings support the bias described by MHRA. Based on our results, apart from vitamin-B12 and possibly cortisol, there may have been no significant influence on clinical decisions as a result of using the old BD SSTII Advance specimen tubes. New BD SSTII Advance tubes and plain tubes give generally comparable results. BD PSTII tubes should not be used for steroid hormone measurements on the Bayer Advia Centaur instrument.

Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth.

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.

PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium.

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.

Expression of the hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density.

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.

Regulation of the expression of the angiogenic enzyme platelet-derived endothelial cell growth factor/thymidine phosphorylase in endometrial isolates by ovarian steroids and cytokines.

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.

Endometrial and myometrial prostaglandin release during the menstrual cycle in relation to menstrual blood loss.

Endometria and myometria were collected at hysterectomy from 42 women with measured menstrual blood loss. Specimens were obtained throughout the menstrual cycle [menstrual (n = 11), follicular (n = 9), early luteal (n = 6), midluteal (n = 9), and late luteal (n = 7)]. In vitro endometrial and myometrial release of prostaglandin F2 alpha (PGF2 alpha), PGE2, and 6-keto-PGF1 alpha were examined using a superfusion technique. In the endometrium, the principal PG produced was PGF2 alpha, while in the myometrium, it was 6-keto-PGF1 alpha. Cyclical changes in PG release were found only in the endometrium, with an increase toward the end of the menstrual cycle and during menstruation. No significant correlation was found between menstrual blood loss (range, 4-840 ml) and PGF2 alpha, PGE2, or 6-keto-PGF1 alpha release in either endometrium or myometrium. Furthermore, there was no correlation between either the PGF2 alpha to PGE2 or the PGF2 alpha to 6-keto-PGF1 alpha ratios and menstrual blood loss.

Steroids and the endometrium.

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.

Identification of t-PA as the major active plasminogen activator in human milk.

Human colostrum and milk contain high levels of plasminogen activator when compared with blood. These levels are inversely related to the duration of lactation and the activity appears to be due to tissue type plasminogen activator present in multiple (65-190 Kd) molecular weight forms.

Examination of certain coagulation factors in menstrual fluid from women with normal blood loss.

Examination of coagulation factors in menstrual fluid collected from women with normal menstrual blood less (less than 80 ml) showed the absence of thrombin generating enzymes. Factor X, pro-thrombin and free thrombin were absent from menstrual fluid. No difference was found between days 1 and 2 of menstruation. Thus activation of coagulation enzymes occurs during normal menstruation though the menstrual blood is characteristically fluid.

Wnt-7a is upregulated by norethisterone in human endometrial epithelial cells: a possible mechanism by which progestogens reduce the risk of estrogen-induced endometrial neoplasia.

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.

Immunohistochemical localization of relaxin, prolactin and prostaglandin synthase in human amnion, chorion and decidua.

Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.

Leukotriene release by endometrium and myometrium throughout the menstrual cycle in dysmenorrhoea and menorrhagia.

Endometrium and myometrium were collected at hysterectomy from 21 women with measured menstrual blood loss. Eight women complained of dysmenorrhea and the remaining 13 had pain-free periods. Specimens were obtained throughout the menstrual cycle (menstrual, n = 5; follicular, n = 4; early luteal, n = 3; mid-luteal, n = 5; late luteal, n = 4). Leukotriene C4, leukotriene D4 and leukotriene E4 release were examined using a short-term incubation technique. Endometrial leukotriene release, which was always significantly greater than myometrial release, changed throughout the menstrual cycle and the highest concentrations were found during menstruation. Endometrial, but not myometrial, leukotriene concentrations were significantly higher in tissues obtained from women with a complaint of dysmenorrhoea compared with those in tissue from pain-free women. No correlation was found between leukotriene release in either endometrium or myometrium and menstrual blood loss (range 15-457 ml).

Leukotriene release by human fetal membranes, placenta and decidua in relation to parturition.

Complete placentas and membranes were obtained from women after uncomplicated singleton pregnancies. Six were collected after labour at term, six after preterm labour and six after elective Caesarean section at term. Leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) release was examined using a short-term incubation technique. Release by amnion was significantly higher than that by the other tissues for the three modes of delivery. Comparison of LTC4, LTD4 and LTE4 release by individual tissues with regard to the type of delivery showed no significant difference.

Consumption of fibrinolytic proteins in menstrual fluid from women with normal menstrual blood loss.

Fibrinolytic proteins in menstrual fluid collected from women with normal menstrual blood loss (less than 80 ml) were found to be completely exhausted. High levels of tissue plasminogen activator, fibrin(ogen) degradation products, and plasmin-inhibitor complexes were present, but little fibrinolytically active plasmin remained. No difference was observed between days 1 and 2 of menstruation.

Human menstruation and eicosanoids.

Menstruation is an event restricted to humans and subhuman primates and in which uterine bleeding and contractions occur. Prostaglandins and leukotrienes are thought to play a major role in the normal process of menstruation as well as in the disorders menorrhagia and dysmenorrhoea.

Oestrogen and progestogen hormone replacement therapy for peri-menopausal and post-menopausal women: weight and body fat distribution.

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)

Variation during the menstrual cycle of immune cell populations in human endometrium.

Morphometric analysis and immunohistology of tissue sections have been used to assess variation, during the normal menstrual cycle, of the bone marrow-derived cell populations in human endometrium. Levels of T cells and macrophages were found to be relatively constant throughout the cycle. In contrast, numbers of large granular lymphocytes, identified as being CD56-positive, were generally low between days 10 and 19, but increased sharply in the latter part of the luteal phase, decreasing again after menstruation. This LGL population is known to be abundant in first trimester pregnancy decidua, and is presumed to play a role in early pregnancy success.

Adrenomedullin and tumour angiogenesis.

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.

Levonorgestrel intrauterine system (LNG-IUS) with conjugated oral equine estrogen: a successful regimen for HRT in perimenopausal women.

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.