Activation of the CDK7 Gene, Coding for the Catalytic Subunit of the Cyclin-Dependent Kinase (CDK)-Activating Kinase (CAK) and General Transcription Factor II H, by the Trans-Activator Protein Tax of Human T-Cell Leukemia Virus Type-1.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

Analysis of Inflammatory Markers in Response to Induction of Reprometabolic Syndrome by a Eucaloric High Fat Diet in Normal Weight Women.

Obesity is associated with chronic low-level inflammation and is known to contribute to metabolic dysfunction and hypogonadotropic hypogonadism, which we have previously termed the 'Reprometabolic Syndrome.' To investigate potential factors involved in obesity-related reproductive endocrine dysfunction, we conducted a secondary analysis of inflammatory markers in a sample of normal weight women exposed to a one-month eucaloric high-fat diet (HFD), which, as reported earlier, induced the relative hypogonadotropic hypogonadism characteristic of Reprometabolic Syndrome. Eighteen healthy women with a BMI between 18.0-24.9 kg/m2 and regular menstrual cycles participated in the study. Frequent blood sampling was performed during the early follicular phase before and after the one-month eucaloric HFD intervention (48% of calories from fat). Serum samples pooled from each participant were analyzed using immunoassay to measure levels of 30 cytokines, interleukins, and chemokines. Differences between pre- and post-HFD intervention measures were examined by one-sample t-tests. Exposure to the eucaloric HFD did not result in changes in body weight. HFD-induction of Reprometabolic Syndrome in normal weight women was associated with a significant elevation only in the anti- inflammatory cytokine IL-10 (p = 0.04). Eotaxin, IL-6 and MIP-1ß also increased in response to the HFD, but not statistically significantly (p = 0.07). Results suggest that the increase in multiple inflammatory markers, typically associated with obesity, are not primary mediators of the relative hypogonadotropic hypogonadism of Reprometabolic Syndrome. Clinical Trials Registration Number: NCT02653092; Date of Registration: January 6, 2016.

Acceptability and Feasibility of Initiating a Low Fat Eating Plan in Reproductive Aged Women with Obesity.

Adverse effects of obesity on reproduction are believed in part due to diet related factors leading to hyperlipidemia and hyperinsulinemia. It is unknown whether administration of a low fat eating plan, regardless of weight loss, will improve reproductive axis function in women with obesity. To develop an acceptable and feasible low fat eating plan for a diverse group of reproductive aged women with obesity. Focus groups to determine preferences and barriers to a planned dietary intervention providing very low fat (22% daily calories from fat) eucaloric food to control fat exposure, but not cause weight loss. Logistics of the intervention and monitoring over three menstrual cycles were discussed. Eighteen women enrolled into 4 different focus groups both live and video, 2 at the University of Colorado and 2 at the Morehouse School of Medicine. All participants expressed interest in implementing a low fat dietary intervention and were further interested in instruction on how to maintain healthy eating habits for future fertility. Provision of ethnically appropriate foods, social support to avoid lapses, and tasty alternatives to high fat foods were considered ideal aspects of a feasible intervention. Incentives and graduated compensation for adherence were considered desirable features. Women with obesity are interested in implementing dietary interventions that may improve their health and fertility. Given the diversity of responses based upon the demographics of our sample, it is important to assess geographical and cultural preferences prior to implementing of a dietary strategy.

Effectiveness of preconception weight loss interventions on fertility in women: a systematic review and meta-analysis.

IMPORTANCE: Weight loss before conception is recommended for women with overweight or obesity to improve fertility outcomes, but evidence supporting this recommendation is mixed. OBJECTIVE: To examine the effectiveness of weight loss interventions using lifestyle modification and/or medication in women with overweight or obesity on pregnancy, live birth, and miscarriage. DATA SOURCES: An electronic search of MEDLINE, Embase, Cochrane Library, including Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials, and Cumulative Index to Nursing and Allied Health Literature was conducted through July 6, 2022, via Wiley. STUDY SELECTION AND SYNTHESIS: Randomized controlled trials examining weight loss interventions through lifestyle and/or medication in women with overweight or obesity planning pregnancy were included. Random-effects meta-analysis was conducted, reporting the risk ratio (RR) for each outcome. Subgroup analyses were conducted by intervention type, type of control group, fertility treatment, intervention length, and body mass index (BMI). MAIN OUTCOME(S): Clinical pregnancy, live birth, and miscarriage events. RESULT(S): A narrative review and meta-analysis were possible for 16 studies for pregnancy (n = 3,588), 13 for live birth (n = 3,329), and 11 for miscarriage (n = 3,248). Women randomized and exposed to a weight loss intervention were more likely to become pregnant (RR = 1.24, 95% CI 1.07-1.44; I2 = 59%) but not to have live birth (RR = 1.19, 95% CI 0.97-1.45; I2 = 69%) or miscarriage (RR = 1.17, 95% CI 0.79-1.74; I2 = 31%) compared with women in control groups. Subgroup analyses revealed women randomized to weight loss interventions lasting 12 weeks or fewer (n = 9, RR = 1.43; 95% CI 1.13-1.83) and women with a BMI ≥ 35 kg/m2 (n = 7, RR = 1.54; 95% CI, 1.18-2.02) were more likely to become pregnant compared with women in the control groups. Miscarriage was higher in intervention groups who underwent fertility treatment (n = 8, RR 1.45; 95% CI 1.07-1.96). CONCLUSION(S): Pregnancy rates were higher in women undergoing preconception weight loss interventions with no impact on live birth or miscarriage rates. Findings do not support one-size-fits-all recommendation for weight loss through lifestyle modification and/or medication in women with overweight or obesity immediately before conception to improve live birth or miscarriage outcomes.

A high-fat eucaloric diet induces reprometabolic syndrome of obesity in normal weight women.

We examined the effects of 1 month of a eucaloric, high-fat (48% of calories) diet (HFD) on gonadotropin secretion in normal-weight women to interrogate the role of free fatty acids and insulin in mediating the relative hypogonadotropic hypogonadism of obesity. Eighteen eumenorrheic women (body mass index [BMI] 18-25 kg/m2) were studied in the early follicular phase of the menstrual cycle before and after exposure to an HFD with frequent blood sampling for luteinizing hormone (LH) and follicle-stimulating hormone (FSH), followed by an assessment of pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Mass spectrometry-based plasma metabolomic analysis was also performed. Paired testing and time-series analysis were performed as appropriate. Mean endogenous LH (unstimulated) was significantly decreased after the HFD (4.3 ± 1.0 vs. 3.8 ± 1.0, P < 0.01); mean unstimulated FSH was not changed. Both LH (10.1 ± 1.0 vs. 7.2 ± 1.0, P < 0.01) and FSH (9.5 ± 1.0 vs. 8.8 ± 1.0, P < 0.01) responses to 75 ng/kg of GnRH were reduced after the HFD. Mean LH pulse amplitude and LH interpulse interval were unaffected by the dietary exposure. Eucaloric HFD exposure did not cause weight change. Plasma metabolomics confirmed adherence with elevation of fasting free fatty acids (especially long-chain mono-, poly-, and highly unsaturated fatty acids) by the last day of the HFD. One-month exposure to an HFD successfully induced key reproductive and metabolic features of reprometabolic syndrome in normal-weight women. These data suggest that dietary factors may underlie the gonadotrope compromise seen in obesity-related subfertility and therapeutic dietary interventions, independent of weight loss, may be possible.

Expanding Roles of the E2F-RB-p53 Pathway in Tumor Suppression.

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

Effects of pulsatile intravenous follicle-stimulating hormone treatment on ovarian function in women with obesity.

OBJECTIVE: To establish conditions for effective hypothalamic suppression in women with normal and high body mass index (BMI) and test the hypothesis that intravenous (IV) administration of pulsatile recombinant follicle-stimulating hormone (rFSH) can overcome the clinically evident dysfunctional pituitary-ovarian axis in women with obesity. DESIGN: Prospective interventional study. SETTING: Academic medical center. PATIENT(S): Twenty-seven normal-weight women and 27 women with obesity, who were eumenorrheic and aged 21-39 years. INTERVENTION(S): Two-day frequent blood sampling study, in early follicular phase, before and after cetrorelix suppression of gonadotropins and exogenous pulsatile IV rFSH administration. MAIN OUTCOME MEASURE(S): Serum inhibin B and estradiol (E2) levels (basal and rFSH stimulated). RESULT(S): A modified gonadotropin-releasing hormone antagonism protocol effectively suppressed production of endogenous gonadotropins in women with normal and high BMIs, providing a model to address the functional role of FSH in the hypothalamic-pituitary-ovarian axis. The IV rFSH treatment resulted in equivalent serum levels and pharmacodynamics in normal-weight women and those with obesity. However, women with obesity exhibited reduced basal levels of inhibin B and E2 and a significantly decreased response to FSH stimulation. The BMI was inversely correlated with serum inhibin B and E2. In spite of this observed deficit in ovarian function, pulsatile IV rFSH treatment in women with obesity resulted in E2 and inhibin B levels comparable with those in normal-weight women, in the absence of exogenous FSH stimulation. CONCLUSION(S): Despite normalization of FSH levels and pulsatility by exogenous IV administration, women with obesity demonstrate ovarian dysfunction with respect to E2 and inhibin B secretion. Pulsatile FSH can partially correct the relative hypogonadotropic hypogonadism of obesity, thereby providing a potential treatment strategy to mitigate some of the adverse effects of high BMI on fertility, assisted reproduction, and pregnancy outcomes. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov #NCT02478775.

The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F.

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool.

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells.

A randomized clinical trial demonstrating cell type specific effects of hyperlipidemia and hyperinsulinemia on pituitary function.

INTRODUCTION: Obesity is characterized by elevated lipids, insulin resistance and relative hypogonadotropic hypogonadism, reducing fertility and increasing risk of pregnancy complications and birth defects. We termed this phenotype 'Reprometabolic Syndrome' and showed that it can be recapitulated by acute infusions of lipid/insulin into healthy, normal weight, eumenorrheic women. Herein, we examined the broader impact of hyperlipidemia and euglycemic hyperinsulinemia on anterior pituitary trophic hormones and their targets. METHODS: Serum FSH, LH, TSH, growth hormone (GH), prolactin (PRL), thyroid hormones (free T4, total T3), cortisol, IGF-1, adiponectin, leptin and creatinine were measured in a secondary analysis of an interventional crossover study of 12 normal weight cycling women who underwent saline and heparin (control) infusion, or a euglycemic insulin infusion with heparin and Intralipid® (lipid/insulin), between days 2-5 in sequential menstrual cycles. RESULTS: In contrast to the decrease in gonadotropins, FSH and LH, infusion of lipid/insulin had no significant effects on other trophic hormones; TSH, PRL or GH. Thyroid hormones (fT4 and total T3), cortisol, IGF-1, adiponectin and creatinine also did not differ between saline or lipid/insulin infusion conditions. Leptin increased in response to lipid/insulin (p<0.02). CONCLUSION: Acute hyperlipidemia and hyperinsulinemia exerted differential, cell type specific effects on the hypothalamic-pituitary-gonadal, adrenal and thyroid axes. Elucidation of mechanisms underlying the selective modulation of pituitary trophic hormones, in response to changes in diet and metabolism, may facilitate therapeutic intervention in obesity-related neuroendocrine and reproductive dysfunction.

Heparin Effects on Serum Gonadotropins.

INTRODUCTION: Studies using lipid infusions to raise fatty acid levels require heparin to release lipoprotein lipase (LPL), thus calling into question the appropriate control infusion for this type of study: saline alone or saline plus heparin. We aimed to evaluate whether the addition of heparin alone, in doses needed to release LPL, would alter circulating free fatty acids (FFAs) and/or affect gonadotropins. MATERIALS AND METHODS: This was a secondary analysis using combined data from eumenorrheic normal-weight women subjected to "control" conditions in 1 of 2 separate studies. In 1 study, participants received saline alone (group 1) as a control, and in the other study participants received saline alone and/or saline plus heparin (groups 2-3) as a control. Both studies performed early follicular phase, frequent blood sampling. FSH and LH were compared across groups and in conditions with and without heparin. Linear mixed models were used to analyze the data. RESULTS: LH did not differ across any of the 3 groups. Estimated means (SE) for FSH differed between groups but this difference was marginal (P = .05) after adjusting for anti-Mullerian hormone and unrelated to heparin infusion (group 1: 4.47 IU/L [SE 1.19], group 2: 8.01 IU/L [SE 1.14], group 3: 7.94 IU/L [SE 1.13]). CONCLUSIONS: Heparin does not exert major effects on gonadotropins when infused in quantities sufficient to release LPL. However, because it can release other vascular membrane-bound proteins, heparin should be considered part of the control infusions in lipid infusion studies where increased FFA levels are the goal.

Inhibiting Wnt/beta-catenin in CTNNB1-mutated endometrial cancer.

The role of ß-catenin/TCF transcriptional activity in endometrial cancer (EC) recurrence is not well understood. We assessed the impact of Wnt/ß-catenin inhibition in EC models. In an analysis of the Cancer Genome Atlas, we confirmed that CTNNB1 mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/ß-catenin pathway activation is associated with recurrence. We studied CTNNB1-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. Dose response curves were determined for 5 Wnt/ß-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). XAV939, Wnt-C59 and PyrPam inhibited function upstream of ß-catenin transcriptional activity and were ineffective at inhibiting cell viability. In contrast, PRI724 and SM04690 indirectly inhibited ß-catenin transcriptional activity and significantly reduced cell viability in CTNNB1-mutant cell lines. Treatment with SM04690 reduced cell viability (Licor Cell stain) in all EC cell lines, but viability was significantly lower in CTNNB1-mutant cell lines (p < 0.01). Mechanistically, SM04690 significantly inhibited proliferation measured via 5'-bromo-2'-deoxyuridine incorporation and reduced T cell factor (TCF) transcriptional activity. HEC1B, HEC1B-S33Y and HEC265 tumor-bearing mice were treated with vehicle or SM04690. Tumors treated with SM04690 had smaller mean volumes than those treated with vehicle (p < 0.001, p = 0.014, p = 0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (p = 0.035, p = 0.024). Targeting the Wnt/ß-catenin pathway in CTNNB1-mutant EC effectively inhibited proliferation and ß-catenin/TCF transcriptional activity and blunted tumor progression in in vivo models. These studies suggest ß-catenin transcriptional inhibitors are effective in EC and particularly in CTNNB1-mutant EC, highlighting a potential therapeutic vulnerability for treatment of CTNNB1-mutant EC.

Gonadotropin response to insulin and lipid infusion reproduces the reprometabolic syndrome of obesity in eumenorrheic lean women: a randomized crossover trial.

OBJECTIVE: To study the reprometabolic syndrome in normal-weight, eumenorrheic women by infusing a combination of insulin and lipid. Women with obesity have been shown to have reduced gonadotropins and impaired luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to gonadotropin-releasing hormone (GnRH). DESIGN: Randomized crossover. SETTING: Academic medical center. PARTICIPANT(S): Fifteen women, median age 32 (interquartile ranged [IQR] 26, 36) years and body mass index 21.9 (IQR 20.2, 22.9) kg/m2 were recruited. INTERVENTION(S): Early follicular phase, 6-hour infusions of insulin (20-40 mU/m2 per minute) and lipid (Intralipid)-insulin/lipid infusion; or saline infusion (controls). The first 4 hours of each study assessed endogenous gonadotropins; at 4 hours, GnRH (75 ng/kg) bolus was administered and sampling continued until 6 hours. MAIN OUTCOME MEASURE(S): Linear mixed model analysis was used to determine differences between insulin/lipid and saline influence on endogenous LH pulse amplitude (primary outcome), mean FSH, and area under the curve (AUC) response to GnRH (secondary outcomes). RESULT(S): Twelve women completed both intended studies and an additional 3 women completed only 1 of the 2 studies. LH pulse amplitude, mean FSH, and both AUC responses to GnRH were reduced by insulin/lipid, mean FSH and AUC for LH were at or near statistical significance. LH response to GnRH was significantly reduced when 1 participant with very high LH and antimullerian hormone levels was excluded. CONCLUSION(S): Acute infusion of insulin/lipid to eumenorrheic, normal-weight women recapitulated the reprometabolic syndrome of obesity. These findings imply that specific circulating factors in obese women contribute to their subfertility and thus may be amenable to discovery and treatment. CLINICAL TRIAL REGISTRATION NUMBER: NCT02653092.

A randomised trial examining inflammatory signaling in acutely induced hyperinsulinemia and hyperlipidemia in normal weight women-the reprometabolic syndrome.

CONTEXT: Obesity, is a state of chronic inflammation, characterized by elevated lipids, insulin resistance and relative hypogonadotropic hypogonadism. We have defined the accompanying decreased Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), ovarian steroids and reduced pituitary response to Gonadotropin-releasing Hormone (GnRH) as Reprometabolic syndrome, a phenotype that can be induced in healthy normal weight women (NWW) by acute infusion of free fatty acids and insulin. OBJECTIVE: To identify potential mediators of insulin and lipid-related reproductive endocrine dysfunction. DESIGN, SETTING, PARTICIPANTS: Secondary analysis of crossover study of eumenorrheic reproductive aged women of normal Body Mass Index (BMI) (<25 kg/m2) at an academic medical center. INTERVENTION: Participants underwent 6-hour infusions of either saline/heparin or insulin plus fatty acids (Intralipid plus heparin), in the early follicular phase of sequential menstrual cycles, in random order. Euglycemia was maintained by glucose infusion. Frequent blood samples were obtained. MAIN OUTCOME MEASURES: Pooled serum from each woman was analyzed for cytokines, interleukins, chemokines, adipokines, Fibroblast Growth Factor-21 (FGF-21) and markers of endoplasmic reticulum (ER) stress (CHOP and GRP78). Wilcoxon signed-rank tests were used to compare results across experimental conditions. RESULTS: Except for Macrophage Inflammatory Protein-1ß (MIP-1ß), no significant differences were observed in serum levels of any of the inflammatory signaling or ER stress markers tested. CONCLUSION: Acute infusion of lipid and insulin, to mimic the metabolic syndrome of obesity, was not associated with an increase in inflammatory markers. These results imply that the endocrine disruption and adverse reproductive outcomes of obesity are not a consequence of the ambient inflammatory environment but may be mediated by direct lipotoxic effects on the hypothalamic-pituitary-ovarian (HPO) axis.

Aromatase Inhibition Ameliorates Decreased LH Output Found in Obese Women.

In obese ovulatory women, serum luteinizing Hormone (LH) and follicle stimulating hormone (FSH) are lowered compared with normal weight women. This relative hypogonadotropic hypogonadism represents a potential etiology for overall decreased fertility in obesity. The objective was to determine if administration of an aromatase inhibitor (AI) to ovulating obese women would normalize LH and FSH by interrupting estradiol negative feedback. Letrozole (2.5-5 mg) was given daily to 22 women, 12 obese and 10 normal weight, for 7 days. On the last day of administration, 8 h of blood sampling was done every 10 min before and after a bolus of GnRH at 4 h. We obtained data from 21 ovulatory women (10 normal weight and 11 obese) who had undergone a similar protocol of frequent blood sampling but no aromatase inhibitors (AI) treatment. Serum LH and FSH levels and pulse characteristics were measured. Treatment with AI only significantly affected obese women. Further, in women with obesity, LH secretion, prior to the GnRH bolus, was significantly higher in AI treated compared with non-treated (p = 0.011). AI treatment doubled LH pulse amplitude in obese women (p = 0.004). In response to aromatase inhibition, LH secretion in ovulatory women with obesity is increased and similar to levels found in untreated normal weight women. The increase in LH pulse amplitude indicates that the AI effect is mediated at the level of the pituitary. Our results suggest that the hypogonadotropic phenotype of simple obesity is subject to modulation by interruption of estradiol negative feedback.

Identification of a novel diagnostic gene expression signature to discriminate uterine leiomyoma from leiomyosarcoma.

Leiomyosarcomas are rare, aggressive tumors, which exhibit a poor prognosis regardless of stage. Pre-operative diagnosis can be difficult as leiomyosarcoma can mimic features of the more common, benign uterine leiomyoma. The goal of this study was to identify specific molecular markers to discriminate between uterine leiomyosarcomas and leiomyomas to facilitate timely, accurate diagnosis and treatment. Gene expression profiles of three leiomyosarcomas, leiomyomas, and normal myometrial tissue samples were analyzed using the Affymetrix Human Gene 1.0 ST Array. GC-robust multiarray average calculation and ANOVA statistical testing were used to identify differentially expressed genes. Sixty genes, with functional roles in tumor progression or suppression, exhibited divergent expression profiles in leiomyosarcomas and leiomyomas, compared to normal myometrium. Differential RNA and protein levels of seven genes, with the most discriminatory expression patterns, were confirmed by RTPCR and immunohistochemistry in an additional 10 leiomyosarcoma and 20 leiomyoma independent samples. CHI3L1, MELK, PRC1, TOP2A, and TPX2 were overexpressed in leiomyosarcomas, while HPGD and TES were overexpressed in leiomyomas. Distinguishing leiomyosarcomas from leiomyomas represents a diagnostic challenge, particularly in the context of minimally invasive surgery. The unique gene expression signatures identified in this study may accurately differentiate between these tumor types at the earliest stage and provides potential prognostic factors and novel therapeutic targets for the treatment of leiomyosarcoma.

Reduction in FSH Throughout the Menstrual Cycle After Omega-3 Fatty Acid Supplementation in Young Normal Weight but not Obese Women.

Dietary fish oil restores ovarian function in subfertile rats, which is thought to be associated with decreased transcription of follicle-stimulating hormone (FSH) ß-subunit. We have previously demonstrated a reduction in early follicular serum FSH levels in normal weight but not obese women after treatment with omega-3 polyunsaturated fatty acids (PUFA). Herein, we report the effect of supplementation with omega-3 PUFA on urinary reproductive hormones across the whole menstrual cycle. This interventional study included 17 eumenorrheic women, aged 24-41 years. One month of daily morning urine was collected before and after 1 month of omega-3 PUFA supplementation with 4 g of eicosapentaenoic acid and docosahexaenoic acid daily. Measurements included urinary FSH, luteinizing hormone (LH) and estrogen and progesterone metabolites, plasma fatty acid composition, and markers of endoplasmic reticulum stress. Compliance with dietary supplementation was verified by significantly reduced ratios of omega-6 to omega-3 PUFA for all subjects after treatment (P < .01). After 1 month of omega-3 PUFA supplementation, urinary FSH was significantly decreased in normal weight, but not obese women, in both follicular and luteal phases (-28.4% and -12.6%, respectively, both P = .04). No significant changes were seen in LH or sex steroids for either weight group. The selective and specific decrease in FSH suggests that omega-3 PUFA supplementation merits further investigation in normal weight women with decreased fertility and/or diminished ovarian reserve.

Differential requirement for dimerization partner DP between E2F-dependent activation of tumor suppressor and growth-related genes.

The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.