Genetic risk factors for endometriosis near estrogen receptor 1 and coexpression of genes in this region in endometrium.

The etiology and pathogenesis of endometriosis are complex with both genetic and environmental factors contributing to disease risk. Genome-wide association studies (GWAS) have identified multiple signals in the estrogen receptor 1 (ESR1) region associated with endometriosis and other reproductive traits and diseases. In addition, candidate gene association studies identified signals in the ESR1 region associated with endometriosis risk suggesting genetic regulation of genes in this region may be important for reproductive health. This study aimed to investigate hormonal and genetic regulation of genes in the ESR1 region in human endometrium. Changes in serum oestradiol and progesterone concentrations and expression of hormone receptors ESR1 and progesterone receptor (PGR) were assessed in endometrial samples from 135 women collected at various stages of the menstrual cycle. Correlation between hormone concentrations, receptor expression and expression of genes in the ESR1 locus was investigated. The effect of endometriosis risk variants on expression of genes in the region was analyzed to identify gene targets. Hormone concentrations and receptor expression varied significantly across the menstrual cycle. Expression of genes in the ESR1 region correlated with progesterone concentration; however, they were more strongly correlated with expression of ESR1 and PGR suggesting coregulation of genes. There was no evidence that endometriosis risk variants directly regulated expression of genes in the region. Limited sample size and cellular heterogeneity in endometrial tissue may impact the ability to detect significant genetic effects on gene expression. Effects of these variants should be validated in a larger dataset and in relevant individual cell types.

Superficial peritoneal endometriotic lesions are histologically diverse and rarely demonstrate menstrual cycle synchronicity with matched eutopic endometrium.

STUDY QUESTION: Do menstrual cycle-dependent changes occur in the histological appearance of superficial peritoneal endometriotic lesions, and are they equivalent to those observed in the eutopic endometrium? SUMMARY ANSWER: Only a small subset of superficial peritoneal endometriotic lesions exhibits some histological features in phase with menstrual cycle-related changes observed in eutopic endometrium. WHAT IS KNOWN ALREADY: Endometriotic lesions are frequently described as implants that follow menstrual cycle-related changes in morphology, as per the eutopic endometrium. This concept has been widely accepted despite the lack of conclusive published evidence. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study of 42 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Patients were a subset selected from a larger endometriosis study being conducted at the Royal Women's Hospital, Melbourne since 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological features of epithelium, stroma and gland morphology were examined in haematoxylin and eosin stained sections of superficial peritoneal endometriotic lesions and matched eutopic endometrium (menstrual: n = 4, proliferative: n = 11, secretory: n = 17, hormone-treated: n = 10). At least two biopsies (average = 4, range = 2-8 biopsies) and a matched endometrial sample were analysed for each patient and results were presented per endometriotic gland profile (n = 1051). Data were analysed using mixed effects logistic regression to account for multiple patients and multiple endometriotic biopsies, each with multiple endometriotic gland profiles. This model also enabled analysis of endometriotic lesions versus eutopic endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: There was considerable inter- and intra-patient variability in the morphology of superficial peritoneal endometriotic lesions. Menstrual cycle-associated changes were only observed for some features in a subset of endometriotic gland profiles. The proportion of endometriotic gland profiles with epithelial mitoses significantly increased in the proliferative phase (18% of gland profiles) relative to the menstrual phase (0% of endometriotic gland profiles) (odds ratios (OR) 9.30; 95% confidence intervals (CI) = 3.71-23.32; P < 0.001). Fewer blood-filled gland lumens were observed in the secretory phase (45% of endometriotic gland profiles) compared to the menstrual phase (67% of endometriotic gland profiles) (OR, 0.30; 95% CI = 0.11-0.79; P = 0.015). The features of the eutopic endometrium analysed in this study did not reflect the results in matched endometriotic lesions (P > 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study focused on features observed in sections of superficial peritoneal lesions and these may differ from features of deep infiltrating endometriosis or ovarian endometriomas. Cycle phases were limited to menstrual, proliferative and secretory phases to allow appropriate statistical modelling. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights heterogeneity in the histological characteristics of superficial peritoneal lesions. It challenges the assumption that lesion morphology consistently reflects menstrual cycle-associated changes. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported in part by National Health and Medical Research Council (NHMRC) project grants GNT1012245, GNT1105321 and GNT1026033 (P.A.W.R., J.E.G. and S.J.H.-C.). There are no competing interests.

Generation of immortalized human endometrial stromal cell lines with different endometriosis risk genotypes.

Endometriotic lesions are composed in part of endometrial-like stromal cells, however, there is a shortage of immortalized human endometrial stromal cultures available for research. As genetic factors play a role in endometriosis risk, it is important that genotype is also incorporated into analysis of pathological mechanisms. Human telomerase reverse transcriptase (hTERT) immortalization (using Lenti-hTERT-green fluorescent protein virus) took place following genotype selection; 13 patients homozygous for either the risk or non-risk 'other' allele for one or more important endometriosis risk single nucleotide polymorphism on chromosome 1p36.12 (rs3820282, rs56318008, rs55938609, rs12037376, rs7521902 or rs12061255). Short tandem repeat DNA profiling validated that donor tissue matched that of the immortalized cell lines and confirmed that cultures were genetically novel. Expression of morphological markers (vimentin and cytokeratin) and key genes of interest (telomerase, estrogen and progesterone receptors and LINC00339) were examined and functional assays for cell proliferation, steroid hormone and inflammatory responses were performed for 7/13 cultures. All endometrial stromal cell lines maintained their fibroblast-like morphology (vimentin-positive) and homozygous endometriosis-risk genotype following introduction of hTERT. Furthermore, the new stromal cultures demonstrated positive and diverse responses to hormones (proliferation and decidualisation changes) and inflammation (dose-dependent response), while maintaining hormone receptor expression. In conclusion, we successfully developed a range of human endometrial stromal cell lines that carry important endometriosis-risk alleles. The wider implications of this approach go beyond advancing endometriosis research; these cell lines will be valuable tools for multiple endometrial pathologies offering a level of genetic and phenotypic diversity not previously available.

Endometrial uNK cell counts do not predict successful implantation in an IVF population.

STUDY QUESTION: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? SUMMARY ANSWER: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF. WHAT IS ALREADY KNOWN: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus.

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17ß (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

A molecular staging model for accurately dating the endometrial biopsy.

Natural variability in menstrual cycle length, coupled with rapid changes in endometrial gene expression, makes it difficult to accurately define and compare different stages of the endometrial cycle. Here we develop and validate a method for precisely determining endometrial cycle stage based on global gene expression. Our 'molecular staging model' reveals significant and remarkably synchronised daily changes in expression for over 3400 endometrial genes throughout the cycle, with the most dramatic changes occurring during the secretory phase. Our study significantly extends existing data on the endometrial transcriptome, and for the first time enables identification of differentially expressed endometrial genes with increasing age and different ethnicities. It also allows reinterpretation of all endometrial RNA-seq and array data that has been published to date. Our molecular staging model will significantly advance understanding of endometrial-related disorders that affect nearly all women at some stage of their lives, such as heavy menstrual bleeding, endometriosis, adenomyosis, and recurrent implantation failure.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus.

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin bA subunit (Inhba) and the inhibin a subunit (Inha) in uterine tissues during early pregnancy (days 1­4, preimplantation) and in response to exogenous 17b-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17b-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus

A Monte Carlo model of synchrotron radiotherapy shows good agreement with experimental dosimetry measurements: Data from the imaging and medical beamline at the Australian Synchrotron.

Experimental measurement of Synchrotron Radiotherapy (SyncRT) doses is challenging, especially for Microbeam Radiotherapy (MRT), which is characterised by very high dynamic ranges with spatial resolutions on the micrometer scale. Monte Carlo (MC) simulation is considered a gold standard for accurate dose calculation in radiotherapy, and is therefore routinely relied upon to produce verification data. We present a MC model for Australian Synchrotron's Imaging and Medical Beamline (IMBL), which is capable of generating accurate dosimetry data to inform and/or verify SyncRT experiments. Our MC model showed excellent agreement with dosimetric measurement for Synchrotron Broadbeam Radiotherapy (SBBR). Our MC model is also the first to achieve validation for MRT, using two methods of dosimetry, to within clinical tolerances of 5% for a 20×20 mm2 field size, except for surface measurements at 5 mm depth, which remained to within good agreement of 7.5%. Our experimental methodology has allowed us to control measurement uncertainties for MRT doses to within 5-6%, which has also not been previously achieved, and provides a confidence which until now has been lacking in MRT validation studies. The MC model is suitable for SyncRT dose calculation of clinically relevant field sizes at the IMBL, and can be extended to include medical beamlines at other Synchrotron facilities as well. The presented MC model will be used as a validation tool for treatment planning dose calculation algorithms, and is an important step towards veterinary SyncRT trials at the Australian Synchrotron.

An RNA spiking method demonstrates that 18S rRNA is regulated by progesterone in the mouse uterus.

Identifying suitable housekeeping genes for quantitative RT-PCR in the uterus is problematic, as this tissue undergoes significant structural and functional alterations during the oestrous cycle and pregnancy in response to circulating hormones. The suitability of 18S rRNA as a housekeeping gene in mouse uterus was investigated by introducing an 'RNA spike' standard into the reverse transcription reaction. 18S rRNA levels increased by Day 4 of pregnancy and after progesterone administration in ovariectomized mice. We conclude that 18S rRNA is not a suitable housekeeping gene for quantitative RT-PCR analysis in progesterone-responsive tissues, and the RNA spiking method provides a suitable alternative.

Endometrial lymphangiogensis.

This article briefly summarises some of the more important recent advances in understanding of lymphangiogenesis, and then reviews current knowledge of the lymphatics and lymphangiogenesis in the endometrium. The recent identification of vascular endothelial growth factor-C (VEGF-C) and VEGF-D, as well as specific lymphatic endothelial cell (LEC) markers such as vascular endothelial growth factor receptor-3 (VEGF-R3), lymphatic endothelial hyaluronan receptor-1 (LYVE-1), podoplanin, and prospero-related homeobox-1 (PROX1), has provided the tools to characterize and investigate lymphatic development and function in a wide range of tissues. There are conflicting reports on the distribution of endometrial lymphatics, with some studies reporting lymphatics in the functional zone of human endometrium, others only in the endometrial basalis, and some reporting none at all. Using immunohistochemical methods we have shown that lymphatic vessels of the functionalis were small and sparsely distributed whereas the basalis lymphatics are larger, more frequent and often closely associated with spiral arterioles. Based on comparisons of serial sections, the majority of lymphatic vessels are positive for CD31 but not FVIII or CD34. By comparing CD31 with D2-40 (labels lymphatic endothelial cells) vessel immunostaining, it was estimated that 13% of the vessel profiles in the functionalis, 43% in the basalis and 28% in the myometrium were lymphatics. The lymphangiogenic growth factor VEGF-C is immunolocalized most prominently in the glandular cells, vascular endothelium and some stromal cells in normal cycling endometrium. There is no difference in staining intensity observed between the basalis and functionalis. VEGF-D is immunolocalized throughout the endometrial and myometrial tissues, with no difference in intensity between endometrial glands and stroma or between the basalis and functionalis across the normal cycle. In conclusion, despite an apparently similar distribution of VEGF-C, VEGF-D and VEGF-R3 in endometrial functionalis and basalis, the lymphatic vascular density is 4-5 times higher in the basalis compared to the functionalis. There is also a close association between some lymphatics in the basalis and the spiral arterioles, thus identifying a potential mechanism for a vascular control feedback loop.

Flexible sigmoidoscopy and whole colonic imaging in the diagnosis of cancer in patients with colorectal symptoms.

BACKGROUND: The aim was to identify the patients with colorectal symptoms most likely to benefit from whole colonic imaging (WCI) to diagnose colorectal cancer and those for whom flexible sigmoidoscopy (FS) may be initially sufficient. METHODS: This prospective observational study (16 years) included 16 433 newly referred patients with symptoms or signs of colorectal cancer. RESULTS: Colorectal cancer was diagnosed in 946 patients (diagnostic yield 5.8 per cent), 815 (86.2 per cent) in the rectum or sigmoid (distal) and 131 (13.8 per cent) in the proximal colon. Some 15 829 patients (96.3 per cent) presented with symptoms alone (without iron deficiency anaemia or abdominal mass). Of 787 cancers in these patients, 750 (95.3 per cent) were distal. The prevalence of proximal cancer above and below the age of 60 years was 0.4 per cent (33 of 8249) and 0.1 per cent (four of 7580) respectively. Of 16 256 patients having FS, 5665 (34.8 per cent) had WCI. Of the other 10 591, five subsequently presented with proximal cancers. FS missed ten (1.3 per cent) of 796 cancers. CONCLUSION: Patients with iron deficiency anaemia or a mass require WCI. However, in patients with symptoms alone, FS detects 95 per cent of cancers, and the diagnostic yield of WCI after FS is very low. Alternative management strategies need to be developed to avoid unnecessary investigations in this low-risk group.

A method of dosimetry for synchrotron microbeam radiation therapy using radiochromic films of different sensitivity.

This paper describes a method of film dosimetry used to measure the peak-to-valley dose ratios for synchrotron microbeam radiation therapy (MRT). Two types of radiochromic film (manufactured by International Specialty Products, NJ, USA) were irradiated in a phantom and also flush against a microbeam collimator (beam width 25 microm, centre-to-centre spacing 200 microm) on beamline BL28 B2 at the SPring-8 synchrotron. Four experiments are reported: (1) the HD-810 and EBT varieties of radiochromic film were used to record 'peak' dose and 'valley' (regions in between peaks) dose, respectively; (2) a stack of HD-810 film sheets was microbeam-irradiated and analysed to investigate a possible dose build-up effect; (3) a very high MRT dose was delivered to HD-810 film to elicit a measurable valley dose to compare with the result obtained using broad beam radiation; (4) the half value layer of the beam with and without the microbeam collimator was measured to investigate the effect of the collimator on the beam quality. The valley dose obtained for films placed flush against the collimator was approximately 0.2% of the peak dose. Within the water phantom, the valley dose had increased to between 0.7 and 1.8% of the peak dose, depending on the depth in the phantom. We also demonstrated, experimentally and by Monte Carlo simulation, that the dose is not maximal on the surface and that there is a dose build-up effect. The microbeam collimator did not make an appreciable difference to the beam quality. The values of the peak-to-valley ratio reported in this paper are higher than those predicted by previously published Monte Carlo simulation papers.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

Eosinophil-Associated Gene Pathways but not Eosinophil Numbers are Differentially Regulated between Synchrotron Microbeam Radiation Treatment and Synchrotron Broad-Beam Treatment by 48 Hours Postirradiation.

Synchrotron microbeam radiation treatment (MRT) is a preclinical radiotherapy technique with considerable clinical promise, although some of the underlying radiobiology of MRT is still not well understood. In recently reported studies, it has been suggested that MRT elicits a different tumor immune profile compared to broad-beam treatment (BB). The aim of this study was to investigate the effects of synchrotron MRT and BB on eosinophil-associated gene pathways and eosinophil numbers within and around the tumor in the acute stage, 48 h postirradiation. Balb/C mice were inoculated with EMT6.5 mouse mammary tumors and irradiated with microbeam radiation (112 and 560 Gy) and broad-beam radiation (5 and 9 Gy) at equivalent doses determined from a previous in vitro study. After tumors were collected 24 and 48 h postirradiation, RNA was extracted and quantitative PCR performed to assess eosinophil-associated gene expression. Immunohistochemistry was performed to detect two known markers of eosinophils: eosinophil-associated ribonucleases (EARs) and eosinophil major basic protein (MBP). We identified five genes associated with eosinophil function and recruitment (Ear11, Ccl24, Ccl6, Ccl9 and Ccl11) and all of them, except Ccl11, were differentially regulated in synchrotron microbeam-irradiated tumors compared to broad-beam-irradiated tumors. However, immunohistochemical localization demonstrated no significant differences in the number of EAR- and MBP-positive eosinophils infiltrating the primary tumor after MRT compared to BB. In conclusion, our work demonstrates that the effects of MRT on eosinophil-related gene pathways are different from broad-beam radiation treatment at doses previously demonstrated to be equivalent in an in vitro study. However, a comparison of the microenvironments of tumors, which received MRT and BB, 48 h after exposure showed no difference between them with respect to eosinophil accumulation. These findings contribute to our understanding of the role of differential effects of MRT on the tumor immune response.

Differences between the pre-menopausal and post-menopausal uterine fibroid vasculature.

OBJECTIVES: To quantitatively examine differences in microvascular density between fibroid and myometrial tissue from fibroid uteri removed at hysterectomy, both before and after the menopause, and with hormone replacement therapy. METHODS: Factor VIII immunostaining of formalin fixed tissues was used to identify blood vessels, and the vessels counted by an investigator blinded to tissue type or menopausal status. RESULTS: The mean myometrial: fibroid MVD ratio was 2.38 higher in the post-menopausal group (95% CI: 0.12, 4.65, p=0.0474) than in the pre-menopausal group, with the hormone therapy (HT)-using post-menopausal group lying in between. An increase in microvascular density in the myometrium after the menopause was responsible for most of the change in ratios seen between the pre and post-menopausal pairs. There was a trend to increasing myometrial MVD with increasing number of years post-menopause. CONCLUSIONS: Myometrial microvascular density increases markedly after the menopause, while fibroid microvascular density does not alter. Thus, the difference between myometrial and fibroid vasculature becomes greater after the menopause. The implications of this for the treatment of fibroids in post-menopausal women is discussed.

Using chlamydia positivity to estimate prevalence: evidence from the Chlamydia Screening Pilot in England.

Studies have suggested that positivity can be used to estimate the prevalence of Chlamydia trachomatis in large-scale chlamydia screening programmes. A recent pilot of opportunistic screening in England estimated that the prevalence among 16-24-year-old women in Portsmouth and Wirral was 9.8% and 11.2%, respectively. This study assessed the continued validity of positivity as an approximate for prevalence. We re-analysed data from the Chlamydia Screening Pilot to estimate positivity,calculated as total positive tests divided by total tests, and compared these estimates with the previously reported prevalence, measured as the number of women testing positive divided by the total number of women screened. Overall positivity was 9.4% in Portsmouth and 11.0% in the Wirral; these estimates were not statistically different from prevalence, regardless of health-care setting, age group or symptoms. We conclude that positivity can be used as a proxy for prevalence.

What a test for recent infection might reveal about HIV incidence in England and Wales.

BACKGROUND: A laboratory method has been developed that detects recent HIV infection and allows incidence to be estimated by testing single stored antibody-positive specimens. A theoretical exploration of the method's surveillance utility was carried out. METHODS: Using various data sources, HIV incidence rates were postulated. The confidence intervals (CI) for these postulated incidences were calculated using the expected number of recent infections for each postulated incidence, the actual number tested for HIV, and the known number of HIV-1 positives. A test for trend was used to determine when an important change in incidence could be recognized. RESULTS: If the incidence was 5% per annum (p.a.) in homosexual/bisexual men attending sexually transmitted diseases (STD) clinics in London, 64 recent infections would be expected in the 392 HIV-seropositive specimens and, if observed, would result in a 95% CI of 3.1-7.9% p.a. for the incidence rate. An incidence of 1% p.a. in pregnant women would be most unlikely as this would require detection of 193 recent infections, 26 more than the total 167 HIV-seropositive specimens found in 1997. In African women attending STD clinics in London, 30% of prevalent infections would be classified as recent if the incidence was 5% p.a. Further, if the incidence in homosexual/bisexual men were to fall by 50% over 3 years, a decrease of this magnitude would be recognized as significant within 2 years. CONCLUSIONS: The detuned assay will increase the information from HIV serosurveys even where prevalence and incidence are relatively low. Existing surveillance systems should be redesigned to take full advantage of the method.

United Kingdom AIDS survival in adults.

OBJECTIVE: To examine the impact of age, year and region of AIDS diagnosis on early (up to 12 months) and late survival of AIDS patients in different exposure categories; to describe the hazard pattern from 12 months after AIDS diagnosis. PATIENTS: A total of 4577 UK adult AIDS diagnoses to the end of 1991 in men who have sex with men, 273 AIDS cases in injecting drug users, 411 AIDS patients infected by blood products, and 535 other adult AIDS cases, mainly ascribed to heterosexual transmission. Deaths have been recorded for 4739 of these 5796 AIDS patients. RESULTS: The influence of calendar year and region of AIDS diagnosis on survival were short-term, for the most part operative only within the first year of follow-up. The monthly death-rate was roughly constant from 12 to 48 months post-AIDS [pooled estimate, 0.055 with 95% confidence interval (CI), 0.053-0.057] but was more than halved for 4-year survivors (pooled estimate, 0.022; 95% CI, 0.017-0.027). About 7% of AIDS cases diagnosed in 1990-1991 survive for at least 48 months. Survival after AIDS diagnosis shortens with advancing age at AIDS diagnosis: the relative hazard per decade of age (1.35; 95% CI, 1.29-1.41 in the first year after AIDS) is significantly greater (P < 0.001) in the first year after AIDS diagnosis than from 12 to 48 months (1.19; 95% CI, 1.13-1.25 in the second epoch). CONCLUSIONS: The influence of covariates, including age, is strongest in the first year of follow-up after AIDS diagnosis. Monthly death-rate is roughly constant at 0.055 from 12 to 48 months post-AIDS and at 0.022 thereafter.