Lessons from KEEPS: the Kronos Early Estrogen Prevention Study.

The Kronos Early Estrogen Prevention Study (KEEPS) was a randomized, double-blind, placebo-controlled trial designed to determine the effects of hormone treatments (menopausal hormone treatments [MHTs]) on the progression of carotid intima-medial thickness (CIMT) in recently menopausal women. Participants less than 3 years from menopause and without a history of overt cardiovascular disease (CVD), defined as no clinical CVD events and coronary artery calcium < 50 Agatston units, received either oral conjugated equine estrogens (0.45 mg/day) or transdermal 17ß-estradiol (50 µg/day), both with progesterone (200 mg/day for 12 days/month), or placebo pills and patches for 4 years. Although MHT did not decrease the age-related increase in CIMT, KEEPS provided other important insights about MHT effects. Both MHTs versus placebo reduced the severity of menopausal symptoms and maintained bone density, but differed in efficacy regarding mood/anxiety, sleep, sexual function, and deposition of ß-amyloid in the brain. Additionally, genetic variants in enzymes for metabolism and uptake of estrogen affected the efficacy of MHT for some aspects of symptom relief. KEEPS provides important information for use of MHT in clinical practice, including type, dose, and mode of delivery of MHT recently after menopause, and how genetic variants in hormone metabolism may affect MHT efficacy on specific outcomes.

Characterization of cell fusion in an experimental mouse model of endometriosis†.

Cell fusion is involved in the development of some adult organs, is implicated in the pathogenesis of specific types of cancer, and is known to participate in repair/regeneration processes mediated by bone-marrow-derived cells (BMDCs). Endometriosis is a disease characterized by growth of functional endometrial tissue outside of the uterine cavity. Endometriosis shares some molecular properties with cancer and BMDCs home to endometriosis lesions in a mouse model. Our objective was to determine if cell fusion can occur in endometriosis and establish whether bone-marrow-derived cells participate in cell fusion events in lesions. We employed a Cre-Lox system to identify cell fusion events in a mouse model of endometriosis. Fused cells were detected in endometriotic lesions, albeit at a low frequency (∼1 in 400 cells), localized to the stromal compartment, and displayed restricted proliferation. Using 5-fluorouracil-based nongonadotoxic bone marrow transplantation model, we demonstrate that bone marrow cells represent a principal cell source for fusion events in lesions. Cell fusion progeny uniformly lacked expression of selected markers of hematopoietic, endothelial, and epithelial markers, though they expressed the mesenchymal/stromal markers Sca-1 and CD29. This study is the first to describe the phenomenon of cell fusion in endometriosis and points to a mesenchymal population derived from cell fusion events with limited proliferative activity, properties previously attributed to endometrial stem cells. Their putative role in the pathogenesis of the disease remains to be elucidated.

Apparent lack of efficacy of toltrazuril against Eimeria species affecting brown kiwi (Apteryx mantelli) at a captive rearing facility.

AIM: To assess the efficacy of toltrazuril against the Eimeria spp. affecting brown kiwi (Apteryx mantelli). METHODS: Droppings were collected from three brown kiwi, aged <6 months old, at a captive rearing facility in the North Island of New Zealand, between 22 February and 20 April 2017, on 14 sampling dates. Only droppings (n=30) that were excreted between 03:00 and 07:00, as determined using video surveillance, were included for analysis, reflecting the peak time for shedding of coccidial oocysts for brown kiwi. Oocysts were quantified in each sample and Eimeria species identified on the basis of oocyst morphology. All samples were collected between 2 and 10 days after the birds had been treated with 25 mg/kg toltrazuril. RESULTS: Eimeria spp. oocysts were identified in 28/30 individual samples and on 14/14 sampling dates. Oocyst counts varied from 0 to 328,080 oocysts per gram (opg), and at least one oocyst count >10,000 opg was measured on 12/14 sampling dates. Three species of Eimeria were observed, with Eimeria apteryxii and E. kiwii most commonly encountered, whereas only one sample contained E. paraurii. CONCLUSIONS AND CLINICAL RELEVANCE: In the three birds monitored at this research site, there was a high abundance of E. apteryxii and E. kiwii oocysts in droppings despite recent administration of toltrazuril. These results suggest that the populations of Eimeria spp. affecting brown kiwi at this location appear to possess an ability to survive exposure to toltrazuril. Toltrazuril is widely used at captive rearing facilities to limit the effects of coccidiosis in juvenile kiwi. If a lack of efficacy is confirmed, it will be necessary to investigate alternative treatment regimens alongside broader environmental management strategies.

The circadian variation of oocyst shedding of Eimeria spp. affecting brown kiwi (Apteryx mantelli).

Captive rearing of wild brown kiwi (Apteryx mantelli) is widely carried out to assist in the recovery of this declining species. As a consequence, high densities of immunologically naïve kiwi are commonly housed in semi-captive conditions, with the potential to result in substantial morbidity and mortality from coccidiosis caused by multiple species of Eimeria. Previous research has described circadian variation in oocyst shedding across multiple avian host species. The aim of this research was to describe any circadian variation in oocyst shedding in brown kiwi. Droppings were collected from brown kiwi (n = 4) at a single captive rearing facility using video surveillance to determine the time of excretion, and oocyst counts were undertaken. Results show that two of the Eimeria spp. affecting brown kiwi exhibit a peak in oocyst shedding between 03.00 and 07.00 with few or no oocysts shed between 08.00 and midnight. These results are not able to be explained by the current hypotheses theorising the evolutionary forces behind the development of this adaptive trait. Our findings increase the current understanding of the biology of the Eimeria spp. affecting brown kiwi and have important implications for the management of captive-reared kiwi, in particular for the accurate interpretation of faecal oocyst counts.

Increasing sensitivity in determining chemical shifts in one dimensional Lorentzian NMR spectra.

An algorithm is presented for one-dimensional NMR systems that employs nonlinear, non-Fourier methods to convert noisy time-dependent free induction decay (FID) data to a denoised frequency spectrum that gives reliable chemical shifts and coupling constants when the spectrum is Lorentzian. It is formulated in a way that increases frequency sensitivity and resolution and, for nuclei of low natural abundance, potentially avoids enrichment totally or in part. The algorithm should also be of use in analytical chemistry where enrichment is not possible. In effect, the useful limit of detection is significantly lowered. The algorithm uses new "phasing" and "feature stability upon accumulation" methods to reliably separate signal from noise at low signal-to-noise ratios where the Fourier spectrum requires many more transients to be definitive as to what is signal and what is noise. The long-standing problem of "false features" that plagued many prior attempts to employ nonlinear methods is thereby resolved for Lorentzian spectra. Examples are reported, and the limitations of the algorithm are discussed.

Medical management of endometriosis: emerging evidence linking inflammation to disease pathophysiology.

Progesterone action normally mediates the balance between anti-inflammatory and proinflammatory processes throughout the female reproductive tract. However, in women with endometriosis, endometrial progesterone resistance, characterized by alterations in progesterone responsive gene and protein expression, is now considered a central element in disease pathophysiology. Recent studies additionally suggest that the peritoneal microenvironment of endometriosis patients exhibits altered physiological characteristics that may further promote inflammation-driven disease development and progression. Within this review, we summarize our current understanding of the pathogenesis of endometriosis with an emphasis on the role that inflammation plays in generating not only the progesterone-resistant eutopic endometrium but also a peritoneal microenvironment that may contribute significantly to disease establishment. Viewing endometriosis from the emerging perspective that a progesterone resistant endometrium and an immunologically compromised peritoneal microenvironment are biologically linked risk factors for disease development provides a novel mechanistic framework to identify new therapeutic targets for appropriate medical management.

HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium.

Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.

Metformin alters DNA methylation genome-wide via the H19/SAHH axis.

The molecular mechanisms underlying the antineoplastic properties of metformin, a first-line drug for type 2 diabetes, remain elusive. Here we report that metformin induces genome-wide alterations in DNA methylation by modulating the activity of S-adenosylhomocysteine hydrolase (SAHH). Exposing cancer cells to metformin leads to hypermethylation of tumor-promoting pathway genes and concomitant inhibition of cell proliferation. Metformin acts by upregulating microRNA let-7 through AMPK activation, leading to degradation of H19 long noncoding RNA, which normally binds to and inactivates SAHH. H19 knockdown activates SAHH, enabling DNA methyltransferase 3B to methylate a subset of genes. This metformin-induced H19 repression and alteration of gene methylation are recapitulated in endometrial cancer tissue samples obtained from patients treated with antidiabetic doses of metformin. Our findings unveil a novel mechanism of action for the drug metformin with implications for the molecular basis of epigenetic dysregulation in cancer. This novel mechanism of action also may be occurring in normal cells.

Efficient liposome-mediated gene transfection and expression in the intact human uterus.

Although gene therapy has been used for correction of metabolic defects in diseases such as cystic fibrosis, as adjuvant treatment in cancer, and in the treatment of infectious diseases, there has been no report of gene transfer to the intact female reproductive tract. We assessed the ability to transfect the human uterus ex vivo and thereby evaluate the applicability of gene therapy to gynecology. The uterine lumen was accessed transcervically, using an intrauterine insemination catheter. pcDNA3.1 plasmid containing the Escherichia coli lacZ reporter gene was delivered to each uterus via liposome-mediated transfection. Control uteri were transfected with empty pcDNA3.1. Immunohistochemical analysis revealed beta-galactosidase expression in the lacZ-treated uteri in endometrial epithelial cells, endometrial stromal cells, and myometrium to a depth of 1.75 cm from the endometrial-myometrial junction. Highest expression was seen in endometrial glandular epithelial cells, with significant expression in the stroma and adjacent myometrium. Each of these cell types in the control uteri showed no beta-galactosidase expression. Successful gene transfection and expression in the intact human uterus can be accomplished easily, rapidly, and efficiently. Gene therapy may have wide applicability in the treatment and study of gynecologic disease.

HOXA10 expression is repressed by progesterone in the myometrium: differential tissue-specific regulation of HOX gene expression in the reproductive tract.

HOX genes are essential regulators of development in all multicellular organisms, including humans. We have previously shown that HOXA10 is expressed in the developing uterus and later in the adult human endometrium. HOX genes regulate endometrial development in response to sex steroids. Here, we demonstrate that HOXA10 is expressed in the myometrium as well. In situ hybridization reveals abundant HOXA10 expression, and Northern analysis demonstrates differential HOX gene expression in the myometrium throughout the menstrual cycle. HOXA10 expression decreases in the midsecretory phase, coinciding with high serum progesterone levels. Treatment of primary myometrial cell cultures with progesterone decreases HOXA10 expression in vitro-paralleling the expression seen in vivo. Despite the presence of progesterone receptors in the endometrium and myometrium, HOXA10 is differentially regulated in each tissue by progesterone. HOXA10 expression is induced in the stroma and decreased in the myometrium by progesterone. The differential tissue-specific response of this gene in response to progesterone is likely mediated by sex steroid receptor coactivators or corepressors. Decreased myometrial expression of developmental regulatory genes such as HOXA10 in the nonpregnant uterus may dedifferentiate the myometrium and allow growth in preparation for pregnancy.

Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium.

Under the influence of sex steroids, human endometrium undergoes sequential development in preparation for implantation. Hoxa11 is essential for implantation in the mouse. Here we describe a potential role for HOXA11 in human endometrial development and implantation. Northern analysis demonstrates that HOXA11 is expressed in a menstrual cycle phase-dependent fashion in adult human endometrium. HOXA11 messenger RNA levels dramatically increase at the time of implantation and remain increased in pregnancy. In vitro, HOXA11 expression is increased in response to estrogen or progesterone. There is a dose-responsive increase over the physiologic range of progesterone concentration. Pretreatment with Cyclohexamide does not decrease the response to estrogen. Steroids are novel regulators HOX gene expression. The spatial and temporal pattern of HOXA11 expression in the human endometrium suggests a role in endometrial development, implantation, and maintenance of pregnancy.

Interleukin-8 in the human endometrium.

We have previously shown that interleukin-8 (IL-8), a cytokine with neutrophil chemotactic/activating and T cell chemotactic activity, is produced by human endometrial stromal and glandular cells in culture. The present study investigated the temporal and spatial expression of IL-8 messenger ribonucleic acid (mRNA) and protein in the human endometrium. Endometrial tissue (n = 52) was obtained from human uteri after hysterectomy conducted for reasons other than endometrial disease or from endometrial biopsies. The day of the menstrual cycle was established from women's menstrual history and was confirmed by histology. Half of the tissues (n = 26) were snap-frozen in liquid nitrogen, cellular RNA was extracted, and Northern blots were hybridized with a riboprobe complementary to a specific sequence of IL-8 mRNA. The remaining tissues (n = 26) were processed for frozen sections, and immunohistochemistry was performed using mouse antihuman IL-8 antibody. Comparison of IL-8 mRNA levels throughout the menstrual cycle revealed that late secretory and early to midproliferative phase IL-8 expression was significantly greater than midcycle expression (P < 0.02). Analysis of the IL-8 immunohistochemistry revealed that IL-8 protein is found in the surface epithelium and glands throughout the menstrual cycle. There was no detectable immunoreactive IL-8 in the stromal cells. We conclude that IL-8 is produced in the human endometrium in vivo, and the variations of IL-8 mRNA throughout the menstrual cycle suggest that sex hormones may regulate its gene expression. We speculate that IL-8 may modulate the timely recruitment of neutrophils and lymphocytes into the endometrium.

Cholinergic blockade effects on spatial integration versus cue discrimination performance.

A within-subjects investigation was conducted to determine the effects of central versus peripheral cholinergic blockade in animals tested either on a spatial integration task in which the possibility of rule learning was also available or on a visual discrimination task in which the daily location of food was marked by a distinctive visual stimulus pattern. All testing was conducted on the Maier three-table apparatus. It was found that the only effect of the peripheral cholinergic blockade on the performance of either task group was to produce a decrease in exploratory behavior. In contrast, central cholinergic blockade markedly impaired spatial integration performance; however, it did not impair the ability of animals in rule learning or visual discrimination learning. It was also found that central cholinergic blockade impaired the animal's tendency to enter all tables before reentering a given table during the exploratory phase of the daily session. This finding was interpreted as reflecting an impairment of working memory for spatial information, rather than a general impairment in working memory, and this interpretation was applied to the explanation of the deficit in the spatial integration performance.

The role of HOX genes in the development and function of the female reproductive tract.

HOX genes are a family of regulatory genes that encode transcription factors and are essential during embryonic development. These genes are highly conserved between species such that all metazoans possess a common genetic system for embryonic patterning. This system is conserved in the reproductive tract, where HOX genes are involved in the development of the müllerian system. The reproductive tract is unusual in that HOX genes continue to be expressed in the adult. HOX genes are essential both for appropriate reproductive tract development and for adult function. This article reviews the role of HOX genes in the development of the reproductive tract and the effect of HOX gene mutations on the development of the reproductive tract in both mice and humans. It then reviews the role and regulation of HOX genes in the adult function of the reproductive tract, specifically evidence that HOX genes are important for human endometrial development and receptivity.

Implantation in the human: the role of HOX genes.

HOX genes are transcription factors that are essential for the proper development of the müllerian tract in the embryonic period. It has been discovered that HOX genes are expressed in the adult uterus. Two of them, Hoxa10 and Hoxa11, have been demonstrated to be necessary for uterine receptivity and implantation in mice. Recent evidence also suggests such a role for HOX genes in humans. They are likely to be essential regulators of endometrial development in preparation for implantation. This article reviews the role of the HOX genes in the reproductive tract, their patterns of expression and regulation, the outcome of deficient HOX gene expression, and their potential mechanisms of action. The process of implantation is complex, and many molecular markers have been found expressed at high levels in the endometrium in the peri-implantation window. Targeted disruption has revealed that most of these molecules are redundant and not essential for implantation. The importance of Hox genes in this process has been well documented, and they remain one of the few well-characterized molecules necessary for implantation.