Margaret E. Wierman

Association of Testosterone Therapy With Mortality, Myocardial Infarction, and Stroke in Men With Low Testosterone Levels

IMPORTANCE Rates of testosterone therapy are increasing and the effects of testosterone therapy on cardiovascular outcomes and mortality are unknown. A recent randomized clinical trial of testosterone therapy in men with a high prevalence of cardiovascular diseases was stopped prematurely due to adverse cardiovascular events raising concerns about testosterone therapy safety. OBJECTIVES To assess the association between testosterone therapy and all-cause mortality, myocardial infarction (MI), or stroke among male veterans and to determine whether this association is modified by underlying coronary artery disease. DESIGN, SETTING, AND PATIENTS A retrospective national cohort study of men with low testosterone levels (<300 ng/dL) who underwent coronary angiography in the Veterans Affairs (VA) system between 2005 and 2011. MAIN OUTCOMES AND MEASURES Primary outcome was a composite of all-cause mortality, MI, and ischemic stroke. RESULTS Of the 8709 men with a total testosterone level lower than 300 ng/dL, 1223 patients started testosterone therapy after a median of 531 days following coronary angiography. Of the 1710 outcome events, 748 men died, 443 had MIs, and 519 had strokes. Of 7486 patients not receiving testosterone therapy, 681 died, 420 had MIs, and 486 had strokes. Among 1223 patients receiving testosterone therapy, 67 died, 23 had MIs, and 33 had strokes. At 3 years after coronary angiography, the Kaplan-Meier estimated cumulative percentages with events were 19.9%in the no testosterone therapy group vs 25.7%in the testosterone therapy group,with an absolute risk difference of 5.8%(95%CI, -1.4%to 13.1%) [corrected].The Kaplan-Meier estimated cumulative percentages with events among the no testosterone therapy group vs testosterone therapy group at 1 year after coronary angiography were 10.1% vs 11.3%; at 2 years, 15.4% vs 18.5%; and at 3 years, 19.9% vs 25.7 [corrected].There was no significant difference in the effect size of testosterone therapy among those with and without coronary artery disease (test for interaction, P = .41). CONCLUSIONS AND RELEVANCE Among a cohort of men in the VA health care system who underwent coronary angiography and had a low serum testosterone level, the use of testosterone therapy was associated with increased risk of adverse outcomes. These findings may inform the discussion about the potential risks of testosterone therapy.

Loss of The Retinoblastoma Tumor-Suppressor Gene in Parathyroid Carcinoma

BACKGROUND The origin and molecular pathogenesis of parathyroid carcinoma are unknown. This life-threatening cause of primary hyperparathyroidism cannot be reliably distinguished from its benign counterpart on the basis of histopathological features alone. Because the PRAD1, or cyclin D1, gene, a cell-cycle regulator, has been implicated in a subgroup of benign parathyroid tumors, we examined the possibility that another cell-cycle regulator with possible functional links to PRAD1, the retinoblastoma tumor-suppressor gene (RB), might be involved in the molecular pathogenesis of parathyroid carcinoma. METHODS Parathyroid carcinomas from 9 patients and adenomas from 21 were studied for evidence of tumor-specific loss of RB gene DNA (allelic loss) by analysis of four DNA polymorphisms and for evidence of altered expression oF RB protein by immunohistochemical staining. RESULTS All of 11 specimens from 5 patients with parathyroid carcinoma and informative DNA patterns and 1 of 19 specimens from 19 patients with parathyroid adenoma and informative DNA patterns lacked an RB allele. Fourteen of 16 specimens (88 percent) from the nine patients with carcinoma had abnormal expression of RB protein (a complete or predominant absence of nuclear staining for the protein). None of the 19 adenomas, including the tumor with loss of an RB allele, had unequivocally abnormal staining for RB protein. CONCLUSIONS Inactivation of the RB gene is common in parathyroid carcinoma and is likely to be an important contributor to its molecular pathogenesis. The presence of such inactivation may help to distinguish benign from malignant parathyroid disease and may have useful diagnostic, prognostic, and therapeutic implications.

Liganded Androgen Receptor Interaction with β-Catenin NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS

A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Δ371–485)) as a bait, β-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-β-catenin demonstrated that FLAG-β-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5α-dihydrotestosterone, FLAG-β-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen α receptors did not translocate FLAG-β-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate β-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3β, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed β-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of β-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles β-catenin to the nucleus and that nuclear interaction of AR with β-catenin may modulate transcriptional activity in androgen target tissues.

Challenges to the Measurement of Estradiol: An Endocrine Society Position Statement

OBJECTIVE The objective of the study was to evaluate the current state of clinical assays for estradiol in the context of their applications. PARTICIPANTS The participants were appointed by the Council of The Endocrine Society and charged with attaining the objective using published data and expert opinion. EVIDENCE Data were gathered from published sources via online databases (principally PubMed, Ovid MEDLINE, Google Scholar), and the clinical and laboratory experience of the participants. CONSENSUS PROCESS The statement was an effort of the committee and was reviewed by each member. The Clinical Affairs Committee, the Council of The Endocrine Society, and JCEM reviewers reviewed the manuscript and made recommendations. CONCLUSIONS The measurement of estradiol in biological fluids is important in human biology from cradle to grave. In addition to its centrality in sexual development, it has significant effects on skin, blood vessels, bone, muscle, coagulation, hepatic cells, adipose tissue, the kidney, the gastrointestinal tract, brain, lung, and pancreas. Alterations in its plasma concentration have been implicated in coronary artery disease, stroke, and breast cancer. Although modern immunoassays and liquid chromatography/tandem mass spectrometry-based methods for estradiol are reasonably well suited to the diagnosis and management of infertility (nonetheless, imprecision and method-to-method differences remain problematic), the very low concentrations that appear to be crucial in nonreproductive tissues are a separate and more difficult issue. Such levels of estradiol are too low to be routinely measured accurately or precisely, and further evolution of analytical methods and the way in which estradiol is standardized is needed.

Novel Mechanism for Gonadotropin-Releasing Hormone Neuronal Migration Involving Gas6/Ark Signaling to p38 Mitogen-Activated Protein Kinase

ABSTRACT Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive axis. Normal sexual maturation depends on the migration of GnRH neurons from the olfactory placode to the hypothalamus during development. Previously, we showed restricted expression of the membrane receptor adhesion-related kinase (Ark) in immortalized cell lines derived from migratory but not postmigratory GnRH neurons. In addition, Ark and GnRH transcripts were detected along the GnRH neuron migratory route in the E13 mouse cribriform plate. In the present study, we examined the role of Ark and its ligand, Gas6 (encoded by growth arrest-specific gene 6), in GnRH neuron migration. Gas6 stimulated lamellipodial extension, membrane ruffling, and chemotaxis of immortalized NLT GnRH neuronal cells via the Ark receptor. Gas6/Ark signaling promoted activation of the Rho family GTPase Rac, and adenoviral-mediated expression of dominant negative N17Rac abolished Gas6/Ark-induced actin cytoskeletal reorganization and migration of GnRH neuronal cells. In addition, p38 MAPK was activated downstream of Ark and Rac, and inhibition of p38 MAPK with either SB203580 or adenoviral dominant negative p38α also blocked Gas6/Ark-mediated migration. Finally, downstream of Rac and p38 mitogen-activated protein kinase (MAPK), Gas6/Ark signaling promoted activation of MAPK-activated protein kinase 2 and induced phosphorylation of HSP25, a known regulator of cortical actin remodeling. The data are the first to demonstrate a migratory signaling pathway downstream of Ark/Axl family receptors and suggest a previously unidentified role for p38 MAPK in neuronal migration. Furthermore, these studies support a potential role for Ark in the regulation of GnRH neuronal migration.

Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat.

Follicle-stimulating hormone (FSH) beta, luteinizing hormone (LH) beta, and alpha subunit messenger RNA (mRNA) levels were examined in rats after castration and sex-steroid replacement. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta complementary DNA (cDNA). Rat FSH beta mRNA is 1.7 kilobase in size. After ovariectomy, female FSH beta mRNA levels increased fourfold, whereas those of LH beta and alpha increased twenty- and eightfold, respectively. With estradiol, all subunits returned toward normal levels. Male LH beta and alpha mRNA levels rose eight- and fourfold, respectively, 40 d postcastration, but FSH beta mRNA levels increased minimally. After 7 d of testosterone propionate, LH beta and alpha mRNAs declined to normal levels, whereas FSH beta mRNA increased slightly. We conclude that in female rats FSH beta is negatively regulated by gonadal steroids, but to a lesser extent than LH beta or alpha mRNAs, and there is a differential regulation of FSH beta mRNA levels in males as compared with females at the time points examined.

Reexamination of Testosterone, Dihydrotestosterone, Estradiol and Estrone Levels across the Menstrual Cycle and in Postmenopausal Women Measured by Liquid Chromatography Tandem Mass Spectrometry

Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.

REPORTS: Summary of the Recommendations on Sexual Dysfunctions in Women

INTRODUCTION Womens sexual dysfunction includes reduced interest/incentives for sexual engagement, difficulties with becoming subjectively and/or genitally aroused, difficulties in triggering desire during sexual engagement, orgasm disorder, and sexual pain. AIM To update the recommendations published in 2004, from the 2nd International Consultation on Sexual Medicine (ICSM) pertaining to the diagnosis and treatment of womens sexual dysfunctions. METHODS A third international consultation in collaboration with the major sexual medicine associations assembled over 186 multidisciplinary experts from 33 countries into 25 committees. Twenty one experts from six countries contributed to the Recommendations on Sexual Dysfunctions in Women. MAIN OUTCOME MEASURE Expert opinion was based on grading of evidence-based medical literature, widespread internal committee discussion, public presentation, and debate. RESULTS A comprehensive assessment of medical, sexual, and psychosocial history is recommended for diagnosis and management. Indications for general and focused pelvic genital examination are identified. Evidence based recommendations for further revisions of definitions for sexual disorders are given. An evidence based approach to management is provided. Extensive references are provided in the full ICSM reports. CONCLUSIONS There remains a need for more research and scientific reporting on the optimal management of womens sexual dysfunctions including multidisciplinary approaches.

Gonadotropin-releasing hormone (GnRH) neuron migration: Initiation, maintenance and cessation as critical steps to ensure normal reproductive function

GnRH neurons follow a carefully orchestrated journey from their birth in the olfactory placode area. Initially, they migrate along with the vomeronasal nerve into the brain at the cribriform plate, then progress caudally to sites within the hypothalamus where they halt and send projections to the median eminence to activate pituitary gonadotropes. Many factors controlling this precise journey have been elucidated by the silencing or over-expression of candidate genes in mouse models. Importantly, a number of these factors may not only play a role in normal physiology of the hypothalamic-pituitary-gonadal axis but also be mis-expressed to cause human disorders of GnRH deficiency, presenting as a failure to undergo normal pubertal development. This review outlines the current cadre of candidates thought to modulate GnRH neuronal migration. The further elucidation and characterization of these factors that impact GnRH neuron development may shed new light on human reproductive disorders and provide potential targets to develop new pro-fertility or contraceptive agents.

Endocrine Aspects of Women's Sexual Function

INTRODUCTION Endocrine changes during aging as well as endocrine disorders may either directly or indirectly modulate female sexual function by altering sex hormones, or by impacting on vascular, neurogenic, or psychologic factors. AIM To review information on the impact of the hormonal changes associated with aging or those caused by endocrine disorders on female sexual function and current information on the risks and benefits of hormonal treatments. METHODS Committee members outlined topics and reviewed the published literature on endocrine aspects of female sexual function over a 2-year period. Presentation of the recommendations were presented at the International Consultation on Sexual Medicine Paris, France 2009 and revised accordingly. MAIN OUTCOME MEASURES Quality of data published in the literature and recommendations were based on the GRADES system. RESULTS Recommendations and guidelines concerning the role of sex hormones and endocrine disorders in female sexual function were derived. CONCLUSIONS Hormones are only one component of the many factors that contribute to normal sexual function in women. Further research is needed as to the impact of hormones and endocrine disorders on female sexual dysfunction and the benefits and risks of hormonal therapies.