Lack of association between early menopause and non-alcoholic fatty liver disease in postmenopausal women.

Background: The possibility of an association between early menopause and the risk of non-alcoholic fatty liver disease (NAFLD) is as yet unclear.Methods: The subjects consisted of 4354 postmenopausal women who participated in the 2010-2012 Korea National Health and Nutrition Examination Survey. Early, normal, and late menopause were defined as age at menopause <45 years, 45-54 years, and ≥55 years, respectively. NAFLD was defined by a hepatic steatosis index of >36.Results: When compared with normal menopausal women, early or late menopausal women had no significant differences in the odds ratios (ORs) of NAFLD: OR = 1.05, 95% confidence interval (CI), 0.83-1.32 and OR = 1.02, 95% CI, 0.75-1.39, respectively. These results remained similar after adjustment for known risk factors for NAFLD, reproductive factors, and comorbidities. The OR for NAFLD per 1-year increase in age at menopause was 1.01 (95% CI, 0.99-1.03; p = 0.329). The prevalence of advanced fibrosis was 2.1% (95% CI, 0.7-6.4%), 2.2% (95% CI, 1.3-3.8%), and 3.9% (95% CI, 1.2-12.2%) in early, normal, and late menopausal women, respectively.Conclusions: This study provides no evidence for an association of early menopause with NAFLD risk. However, NAFLD-related advanced fibrosis is highly prevalent in postmenopausal women.

Telomeric DNA quantity, DNA damage, and heat shock protein gene expression as physiological stress markers in chickens.

In this longitudinal study with Single Comb White Leghorn chickens, we investigated the effects of stress conditions in birds that were subjected to a high stocking density with feed restrictions on the quantity of telomeric DNA, the rate of DNA damage, and the expression levels of heat shock proteins (HSP) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes. The telomere length and telomere-shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90, and HMGCR genes were measured by quantitative real-time PCR in lymphocytes. The telomere-shortening rate of the lymphocytes was significantly higher in the stress group than in the control. The DNA damage also increased in birds raised under stress conditions, as compared with the control group. The stress conditions had a significant effect on the expressions of HMGCR and HSP90α in lymphocytes but had no significance on HSP70 and HSP90ß in blood. We conclude that the telomere length, especially the telomere-shortening rates, the quantification of total DNA damage, and the expression levels of the HMGCR and HSP90α genes can be used as sensitive physiological stress markers in chickens.

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

GABA- and glycine-like immunoreactivity in axonal endings presynaptic to the vibrissa afferents in the cat trigeminal interpolar nucleus.

The goal of this study was to analyze the synaptic interaction of primary afferents with GABA- and/or glycine-immunopositive presynaptic endings in the cat trigeminal interpolar nucleus (Vi). Fast adapting vibrissa afferents were labeled by intra-axonal injections of horseradish peroxidase. Postembedding immunogold labeling on serially cut ultrathin sections and quantitative ultrastructural analysis of the labeled boutons and their presynaptic endings (p-endings) in the Vi were performed. The majority of p-endings presynaptic to labeled boutons (83%) were immunopositive for both GABA and glycine and 8% were immunopositive for glycine alone. A small fraction of p-endings were immunopositive for GABA alone (4%) or immunonegative for both GABA and glycine (4%). Ultrastructural parameters related to synaptic release, i.e. bouton volume, mitochondrial volume, and active zone area, were significantly larger in the labeled boutons of primary afferents than in the p-endings. The volume of labeled boutons was positively correlated with the number of the postsynaptic dendrites and p-endings. In addition, fairly large-sized labeled boutons and p-endings were frequently observed in the Vi. These results reveal that large majority of vibrissa afferents in the Vi are presynaptically modulated by interneurons immunopositive for both GABA and glycine, and suggest that the Vi plays a distinct role in the processing of orofacial sensory information, different from that of other trigeminal sensory nuclei.

TRIENNIAL GROWTH AND DEVELOPMENT SYMPOSIUM: Molecular mechanisms related to bovine intramuscular fat deposition in the longissimus muscle.

The intramuscular fat (IMF) content of the LM, also known as marbling, is particularly important in determining the price of beef in Korea, Japan, and the United States. Deposition of IMF is influenced by both genetic (e.g., breed, gender, and genotype) and nongenetic factors (e.g., castration, nutrition, stressors, animal weight, and age). Castration of bulls markedly increases deposition of IMF, resulting in improved beef quality. Here, we present a comparative gene expression approach between bulls and steers. Transcriptomic and proteomic studies have demonstrated that the combined effects of increases in lipogenesis, fatty acid uptake, and fatty acid esterification and decreased lipolysis are associated with increased IMF deposition in the LM. Several peripheral tissues (LM, adipose tissues, and the liver) are involved in lipid metabolism. Therefore, understanding the significance of the tissue network in lipid metabolism is important. Here, we demonstrate that lipid metabolism in LM tissues is crucial for IMF deposition, whereas lipid metabolism in the liver plays only a minor role. Metabolism of body fat and IMF deposition in bovine species has similarities with these processes in metabolic diseases, such as obesity in humans and rodents. Extensive studies on metabolic diseases using epigenome modification (DNA methylation, histone modification, and microRNA), microbial metagenomics, and metabolomics have been performed in humans and rodents, and new findings have been reported using these technologies. The importance of applying "omics" fields (epigenomics, metagenomics, and metabolomics) to the study of IMF deposition in cattle is described. New information on the molecular mechanisms of IMF deposition may be used to design nutritional or genetic methods to manipulate IMF deposition and to modify fatty acid composition in beef cattle. Applying nutrigenomics could maximize the expression of genetic potential of economically important traits (e.g., marbling) in animals.

Systemically expressed soluble Tie2 inhibits intraocular neovascularization.

Retinal and choroidal neovascularization are the most frequent causes of severe and progressive vision loss. Studies have demonstrated that Tie2, an endothelial-specific receptor tyrosine kinase, plays a key role in angiogenesis. In this study, we determined whether adenovirus-mediated gene delivery of extracellular domain of the Tie2 receptor (ExTek) could inhibit experimental retinal and choroidal neovascularization. Immunofluorescence histochemistry with a monoclonal antibody to human Tie2 showed that Tie2 expression is prominent around and within the base of newly formed blood vessels of retinal and choroidal neovascular lesions. A single intramuscular injection of adenovirus expressing ExTek genes achieved plasma levels of ExTek exceeding 500 microg/ml in mice for 10 days (in neonates) and 7 days (in adults). This treatment inhibited retinal neovascularization by 47% (p < 0.05) in a murine model of ischemia-induced retinopathy. The same treatment reduced the incidence and extent of sodium fluorescein leakage from choroidal neovascular lesions by 52% (p < 0.05) and 36% (p < 0.01), respectively, in a laser-induced murine choroidal neovascularization model. The same mice showed a 45% (p < 0.001) reduction of integrated area of the choroidal neovascularization. These findings indicate that Tie2 signaling is a common component of the angiogenic pathway in both retinal and choroidal neovascularization, providing a potentially useful target in the treatment of intraocular neovascular diseases.

Estradiol-17beta biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone.

Granulosa cells isolated from the ovaries of hypophysectomized immature rats synthesize and secrete estradiol-17beta and estrone when grown for 2 days in monolayer culture in a synthetic medium containing testosterone (0.5 muM) and a highly purified follicle-stimulating hormone (FSH) preparation (0.25 mug/ml). Secretion is negligible in the absence of either testosterone or FSH, and a highly purified luteinizing hormone (LH) preparation (0.25 mug/ml) was without significant stimulatory effect. It is concluded that FSH regulates estrogen biosynthesis in granulosa cells of hypophysectomized rats by a specific stimulation of the aromatizing enzyme system.

Stimulatory action of follice-stimulating hormone on estradiol-17 beta secretion by hypophysectomized rat ovaries in organ culture.

Ovaries of rats explanted in organ culture 2-3 days after hypophysectomy at 26 days of age synthesize ans secrete estradiol-17beta into the culture medium at approximately linear rates for 48 h. Addition of testosterone (5 times 10- minus 7M) to the culture medium occasionally caused small increases of up to 50 percent in rate of estradiol production. Significant increases (60-200 percent) in estradiol secretion resulted from addition of two different FSH preparations, in concentrations as low as 0.25 mug/ml in the absence of testosterone. In the presence of testosterone, the same FSH preparation caused much more marked increases in estradiol secretion of up to 900 percent. In the absence of testosterone, two different luteinizing hormone preparations failed to increase estradiol secretion significantly, although in the presence of testosterone, small increases were sometimes observed. It is concluded that FSH regulates estradiol biosynthesis in the hypophysectomized rat ovary by a specific stimulatory action on the aromatizing enzyme system.

17 beta-Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle-stimulating hormone.

Cellular sites and gonadotropic control of human follicular estrogen secretion have been assessed by culturing the theca and granulosa components separately under different hormonal conditions. Granulosa cells from human follicles were grown in chemically defined media containing gonadotropins and/or testosterone (T) for 24 h. The production of 17 beta-estradiol (E2) by cells cultivated in T-free media with or without FSH was very low during the culture period. There was a highly significant increase (P less than 0.001) in E2 production when T alone was added and a more marked increase was consistently noted in the presence of FSH and T. In all cases, hCG failed to exert any significant effect on E2 production by granulosa cells in the presence or absence of T. No treatments examined altered the E2 production of thecal cells during a 24-h culture period and the amounts of E2 released into media were negligible when compared with levels produced by granulosa cells from the same follicles. It is concluded that granulosa cells but not thecal cells are the prime site of follicular estrogen production and that FSH regulates estrogen secretion by nonluteinized granulosa cells of the human follicle.

The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells.

Granulosa and theca cells obtained from patients were isolated and cultivated in a chemically defined medium containing gonadotropins and/or testosterone. Progesterone secretion by granulosa cells was consistently stimulated (2-40-fold) in all 5 patients by the addition of follicle-stimulating hormone (FSH, 0.25 micrograms/ml). In the presence of testosterone (0.5 micro M) alone, progesterone production was stimulated (2-8-fold) in 4 out of the 5 patients and cells of one patient showed a greater response to testosterone than to FSH alone. In 2 of the 5 patients, it was also noted that FSH and testosterone acted in a synergistic manner to stimulate the production of progesterone by granulosa cells. On the other hand, human chorionic gonadotropin (hCG, 1.0 IU/ml) alone failed to exert any significant effect. None of the treatments examined altered the production of progesterone by theca cells. These results suggest a role for FSH and testosterone in regulating progesterone biosynthesis by granulosa cells of the human ovary during follicular development.

Clinical and endocrine response to pulsatile intravenous gonadotropins in refractory anovulation.

We investigated the endocrine response to an experimental protocol in which human menopausal gonadotropin was delivered in pulses every 90-120 minutes by an automated pump. Thirty clomiphene citrate- and gonadotropin-releasing hormone-unresponsive anovulatory women were studied over 107 treatment cycles. In response to episodic intravenous (IV) delivery, pulses of FSH and LH were demonstrable in the circulation. In four World Health Organization (WHO) group I anovulatory women, ovulation occurred in all cycles (N = 8); the pregnancy rate per cycle was 63% and the cumulative pregnancy rate 100%. In 26 WHO group II patients, 42.4% of treatment cycles were in 12 women previously refractory to the intramuscular route of administration; the rate for ovulation was 86% (total of 99 cycles), the pregnancy rate per ovulatory cycle was 14%, and the cumulative pregnancy rate 56%. A mild phlebitis occurred at the site of the IV catheter in 24% of treatment cycles. Intravenous delivery of gonadotropins in small pulses had a dose-sparing effect and was an effective method of treating anovulatory infertile patients refractory to other conventional methods of ovulation induction.

Cleavage of the synaptobrevin/vesicle-associated membrane protein (VAMP) of the mouse brain by the recombinant light chain of Clostridium botulinum type B toxin.

The light chain of Clostridium botulinum type B toxin was expressed in Escherichia coli using the expression vector pEt-3a containing phage T7 promoter. The expressed protein was then purified by DEAE-cellulose and phosphocellulose chromatography and the proteolytic activity of the purified light chain was studied. The purified recombinant light chain cleaved synaptobrevin when mixed with the mouse brain microsome and the proteolytic activity of the light chain was inhibited if a metal chelating agent such as EDTA or 2,2'-dipyridyl was added. The recombinant light chain cleaved synaptobrevin more effectively than the native type B toxin. When the native toxin was trypinized and was reduced with DTT, its proteolytic activity was similar to that of the recombinant light chain.

Prevalence and risk factors for erectile dysfunction in primary care: results of a Korean study.

In order to assess the prevalence and associated factors for erectile dysfunction (ED) in primary care, a cross-sectional study was undertaken by questionnaire distributed to consecutive adult male attendees at 32 family practices. ED was assessed by the Korean five-item version of the International Index of Erectile Function (IIEF-5). In total, 3501 completed questionnaires were available for analysis. The prevalence of ED was severe (IIEF-5 score: 5-9) in 1.6% of cases, moderate (10-13) in 10.2%, mild (14-17) in 24.7%, and normal (18-25) in 63.4%. The prevalence of ED increased with age, lower educational status, heavy job-related physical activity, and lower income. ED prevalence was significantly higher in patients with chronic diseases such as diabetes, depression, and anxiety. These results suggest that the age-adjusted prevalence of ED among Korean men can be estimated as 32.2% (95% CI 30.6-33.7). Low socioeconomic status and several diseases such as diabetes, anxiety, and depression, as well as age, were associated with ED.

The role for the uterine insulin-like growth factor I in early embryonic loss after superovulation in the rat.

OBJECTIVES: To examine possible roles of the insulin-like growth factor (IGF) system in increased early embryonic loss after superovulation. DESIGN: Changes in the uterine IGF system were examined in superovulated rats. Insulin-like growth factor I (IGF-I) was infused to the right uterine horns to mimic enhanced IGF-I actions after superovulation. Uterine luminal fluids were collected after IGF-I infusions and embryos were cultured with uterine luminal fluids. MAIN OUTCOME MEASURES: Steroid hormones, IGF-I, IGF binding protein (IGFBP), and IGF-I receptor levels, developmental rate, and cell numbers of embryos. RESULTS: Elevated IGF-I levels and suppressed IGFBP levels were found from days 1 to 3 of pregnancy after superovulation. Uterine luminal fluids of the IGF-I infusion and superovulation groups impaired embryo development in vitro. Anti-IGF-I antibody infusions after superovulation reversed detrimental effects of superovulation. Dialysis of uterine luminal fluids of the IGF-I infusion and superovulation groups before culture improved embryo development. CONCLUSIONS: Enhanced IGF-I actions in the uterus after superovulation may be responsible for the increase of early embryonic loss. The detrimental factor for embryo development seems a small molecule and is likely a local product of the uterus in which IGF-I actions are enhanced.

The chromosome pattern of embryos derived from tripronuclear zygotes studied by cytogenetic analysis and fluorescence in situ hybridization.

OBJECTIVE: To detect the chromosomal complement of embryos, which developed from tripronuclear zygote, using nonradioactive centromeric probes. DESIGN: The chromosome pattern of embryos developing from tripronuclear zygote, studied by fluorescence in situ hybridization, was compared with that of embryos studied by standard cytogenetic methods. SETTING: These embryos were obtained from superovulated patients undergoing IVF treatment. RESULTS: We have attempted to examine the chromosomal complement of 72 embryos derived from tripronuclear zygotes using both traditional cytogenetic analysis and fluorescence in situ hybridization. Of these 72 embryos, 22 were analyzed with fluorescence in situ hybridization and 50 were analyzed with traditional cytogenetic analysis. For fluorescence in situ hybridization analysis, probes specific for the centromeric regions of chromosomes 1, 16, and X were used, with results being obtained from 18 embryos. One embryo was haploid (5.6%), five were triploid (27.8%), and one was hexaploid (5.6%). Eleven (61%) embryos were mosaic. Traditional cytogenetic analysis could be performed on 25 of 50 embryos. Five (20%) were haploid, one (4%) was diploid, seven (28%) were triploid, one (4%) were tetraploid, and two were hexaploid. Nine (36%) were mosaic. CONCLUSION: These findings indicate that not all tripronuclear human zygotes develop into triploid embryos. This study also demonstrates the usefulness of fluorescence in situ hybridization for preimplantation diagnosis and screening for chromosome abnormalities.

Direct effects of clomiphene citrate on the steroidogenic capability of human granulosa cells.

The effects of clomiphene citrate (CC), 17 beta-estradiol (E2), and human chorionic gonadotropin (hCG) on the accumulation of progesterone (P) and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OHP) in cultured human granulosa cells (GC) were examined. In addition, the metabolism of [4-14C]pregnenolone and accumulation of [4-14C]P in response to CC and E2 were determined. The authors conclude the following: (1) the dose-dependent inhibition of P and 20 alpha-OHP production by CC in GC was not reproduced by E2, (2) hCG abolished these effects of CC, (3) these inhibitory actions of CC were not associated with altered 3 beta-hydroxysteroid dehydrogenase activity nor P catabolism indicating that, (4) these actions by CC on the GC occur at some step(s) during steroidogenesis preceding the formation of pregnenolone. These findings may explain, at least in part, the luteal deficiency experienced by women treated with CC, and they also provide a rationale for the use of hCG supplementation during ovulation induction with CC.

Chromosome analysis of human oocytes failing to fertilize in vitro.

To assess the contribution of chromosome anomalies to the high failure of in vitro fertilization (IVF), 94 unfertilized eggs from 43 women participating in the IVF program were cytogenetically investigated. The mean age of the oocyte donors was 33.6 years. Chromosome karyotypes were obtained in 65 of 94 oocytes: 34 oocytes (52.3%) had a normal haploid chromosome complement; 10 (15.4%) were hypohaploid; 7 (10.8%) were hyperhaploid; 8 were diploid; and 6 were hypodiploid or hyperdiploid (from 36 to 53). Eleven eggs showed prematurely condensed chromosomes of the G1-phase from sperm, as well as a set of maternal metaphase II chromosomes. These results are compared to similar reports in the literature.

The outcome of in vitro fertilization and embryo transfer in women with polycystic ovary syndrome failing to conceive after ovulation induction with exogenous gonadotropins.

OBJECTIVE: To assess the outcome of in vitro fertilization and embryo transfer (IVF-ET) in women with refractory polycystic ovarian syndrome (PCOS). DESIGN: Retrospective case series with an age-matched control group. SETTING: Ovulation induction and IVF programs in a tertiary referral center. PATIENTS AND INTERVENTIONS: Nine patients with PCOS who failed standard ovulation induction treatment (clomiphene citrate plus greater than or equal to 6 ovulatory human menopausal gonadotropin [hMG] cycles) underwent 19 cycles of IVF-ET. Forty age-matched tubal factor patients who completed 40 cycles of IVF-ET served as a control group. OUTCOME MEASURES: Demographic features and IVF-ET cycle characteristics were compared using Student's t-test and Fisher's exact test. RESULTS: Cycles of IVF-ET in patients with PCOS were associated with higher estradiol levels (5,222 versus 4,009 pmol/L), lower hMG requirements (15.8 versus 19.6 vials), greater numbers of oocytes (7.6 versus 5.6), and lower fertilization rates (56% versus 75%) compared with tubal factor cycles (P less than 0.05). However, the number of embryos transferred (3.9 versus 4.0) and the clinical pregnancy rate per embryo transfer (24% versus 25%) did not differ significantly between the two groups. CONCLUSION: These results suggest that conception failure after six or more ovulatory hMG cycles in patients with PCOS does not adversely affect subsequent IVF performance.

A comparison of fructosamine and glycosylated haemoglobin measurements at a diabetic clinic.

Fructosamine or glycosylated haemoglobin (HbA1) measurements are most useful in the diabetic clinic if results are available when the patient is seen, with minimum waiting time. Fructosamine measurements have the advantage of being cheaper and faster to perform on large numbers of patients than HbA1 measurements. In order to assess the acceptability of fructosamine as a complete alternative to HbA1, fructosamine, HbA1 and random plasma glucose measurements were made on all patients attending the diabetic clinic for a 6-week period. Either the fructosamine or the HbA1 result was made available when the patient was seen and the other result was given at the end of the clinic for comment as to whether or not it would have altered management. The clinicians indicated that in 7% of cases, the HbA1 result would have altered management if available when the patient was seen, whereas in 2.5% of cases, the fructosamine result would have altered management. The commonest discrepancy was disproportionate elevation of HbA1 with a normal or near normal fructosamine. For the whole group, 32% had a normal fructosamine but only 16% a normal HbA1. The best overall correlation was between HbA1 and glucose (r = 0.67). Fructosamine correlated less well with both HbA1 (r = 0.55) and glucose (r = 0.51). Thus fructosamine was an acceptable alternative to HbA1 in most cases but caution is required, particularly in patients with persistently normal fructosamine results for whom additional HbA1 checks are advised.

Effects of cannabinoids on progesterone release in cultures of rat luteal cells.

This study investigated the direct effects of tetrahydrocannabinols (THC) on progesterone release by cultured rat luteal cells, as a function of dose and time. During a 24-h incubation, the level of progesterone in the culture medium was decreased by 35% and 60% in the presence of 1 microM 11-OH-delta 9-THC and 8 beta-OH-delta 9-THC, respectively, when compared with control cultures. Dose-response analysis revealed that 8 beta-OH-delta 9-THC inhibited progesterone levels at 0.1 microM but not at lower concentrations. The action of 8 beta-OH-delta 9-THC was rapid in onset and a significant effect could be observed as early as 2 h following the addition of the cannabinoid. While luteinizing hormone (LH, 1 microgram/ml) significantly enhanced progesterone release in the culture medium over the respective control levels, this action of LH was dramatically suppressed by the concomitant presence of 8 beta-OH-delta 9-THC at 2, 4 and 24 h in separate experiments. Moreover, the increase in the level of progesterone in the culture medium induced by 8-bromo-cyclic AMP was also attenuated by the concomitant presence of 8 beta-OH-delta 9-THC in the cultures. These results further substantiate a direct action of cannabinoids on the steroidogenic function of the corpus luteum, and that it involves at least some step(s) distal to the LH-sensitive adenylate cyclase system.