Late-onset hypogonadism (LOH) is a pathological disorder that develops in males over the age of 40 and is diagnosed upon strict criteria that requires that the individual have total serum testosterone (T) below the normal limits as well as three symptoms of sexual dysfunction. Recommended therapy for young males with hypogonadism is testosterone replacement therapy (TRT). Treatment of LOH with TRT has increased significantly in the past several years, but studies showing adverse risks associated with TRT have led to a growing concern about the safety of such a treatment. This systematic review will give an overview of the pathology of LOH, clinical diagnosis of LOH, and comorbidities associated with this dysfunction. Benefits of TRT in elderly hypogonadal men have included improvement in cardiovascular function, reduced all-cause mortality, increased sexual function, increased bone mineral density, improved body composition, increased muscle strength, improved quality of life, and improvement in metabolic parameters. However, risks associated with TRT have included infertility, worsening lipid panel parameters, polycythemia, increased risk of prostate cancer, and in some cases, increased risk of adverse cardiovascular events.

The metabolism of androgenic anabolic steroids has been studied in the racing greyhound. Various drug preparations have been investigated utilising different derivatisation techniques, coupled with gas chromatographic analysis, to enable the identification of key metabolites in canine post administration samples. This has led to an increased understanding of some of the generic routes of steroid metabolism that take place in the greyhound. This valuable information can help to support metabolism studies in the future. The identification of specific metabolites for each compound investigated, has provided a means for controlling the misuse of these compounds, and contributed valuable enhancements to screening protocols utilised in the canine sports drug testing industry. Utilisation of the techniques described, resulted in the identification of specific major metabolites of the anabolic steroid methyltestosterone, namely 17??-methyl-5??- androstan-3??-17??-diol and 17??-methyl-5??-androstan-3??,16??,17??-triol. 16??- hydroxylation was shown to be a major phase I metabolic pathway in the canine along with phase II conjugation with glucuronic acid. Similar results were obtained during the metabolism study of the progestatgenic steroid norethisterone. Several di- and trihydroxy metabolites were detected in the glucuronic acid fraction of the post administration urines from this study. The norethisterone metabolism study also provided some insight, into the area of trace contaminants of pharmaceutical preparations. Low levels of nandrolone metabolites were also detected in the norethisterone post administration urine samples, leading to the discovery that the administered pharmaceutical tablets contained small quantities of nandrolone and 19- norandrostenedione, albeit below FDA approved contaminant levels. Modern methods of drug screening employ such highly sensitive techniques, that they allow for the detection of metabolites of such trace contaminants, following administration of the drug preparation to the greyhound. It is therefore important to have a broad understanding of the metabolism of various drug preparations, both banned and permitted substances alike; as detection of a trace amount of a banned substance metabolite, arising from the administration of a permitted medication, whose iii metabolite profile is unknown, and therefore potentially not detected, could present an interesting case. In conjunction with research into controlling the use of banned substances for the purposes of suppressing oestrus in the greyhound bitch, an investigation into normal/reference levels of endogenous hormones has been carried out. The endogenous steroid levels in a population of 212 greyhound bitches have been studied with a view to establishing a method for the detection of the exogenous administration of the endogenous anabolic steroid testosterone. The major urinary metabolites investigated were epiandrosterone, 5??-androstane-3??,17??-diol and 5??-androstane-3??,17??-diol. Statistical evaluations have been carried out to support the implementation of a suitable threshold for the key testosterone metabolites, namely 5??-androstane-3??,17??-diol and epiandrosterone. The detection of 5??-androstane-3??,17??-diol was found to be a very good indicator of the exogenous administration of testosterone to the greyhound bitch, when compared with the reference population data for this metabolite. However, further statistical/analytical data evaluation was deemed necessary before an absolute threshold could be implemented for this analyte, for the purposes of controlling the misuse of testosterone in the racing greyhound bitch. To support the understanding of endogenous steroid levels in the female greyhound, yet further, the endogenous reproductive steroid profiles were measured throughout the entire oestrus cycle of a cohort of 33 racing bitches. The results of the study clearly indicate a surge in androgen metabolites during the first 7-10 days of the oestrus cycle, in particular epiandrosterone and 5??-androstane-3??,17??-diol. This unique set of data has provided detailed information regarding the fluctuating concentrations of androgen and progesterone metabolites (following ovulation), at key stages of the canine oestrus cycle. The information obtained from this research can be used to support regulatory decisions regarding the misuse of testosterone in the racing greyhound bitch.

Background: Kisspeptins, recently discovered hypothalamic neuropeptides encoded by the KISS1 gene, are essential for normal pubertal development and are modulated by diverse endocrine, metabolic and environmental signals. Exogenous kisspeptin administration potently stimulates LH secretion - by direct action on GnRH neurons while kisspeptin antagonists inhibit pulsatile LH secretion. Human studies of kisspeptin had hitherto used kisspeptin-54 that is cleaved further and the smallest bioactive form is a decapeptide (kisspeptin-10) with a shorter half-life. Kisspeptin-10 is thus putatively more attractive in studies assessing LH pulsatility and is also the basis for the development of antagonists. Unmet clinical needs: Decreased LH pulse frequency is the central pathology in pubertal delay, late-onset male hypogonadism and hypothalamic amenorrhoea. Manipulation of LH pulse frequency also has therapeutic potential in contraception, PCOS and sex-steroid dependant diseases such as endometriosis and prostatic hyperplasia. Hypothesis: That exogenous kisspeptin-10 enhances pulsatile LH secretion in healthy men and in patients with reproductive disorders associated with decreased pulse frequency. Research strategy: A first-in-human dose escalation study of kisspeptin-10 was performed in men and subsequently replicated in women. An intravenous infusion regime was optimised in healthy men and subsequently applied to hypogonadal patients. Specific questions were addressed sequentially as summarised below with key results. Dose escalation study: Question: Does kisspeptin-10 stimulate LH secretion in men? Findings: Six iv bolus doses (0.01 to 3 μg/kg) of GMP kisspeptin-10 and vehicle were administered at least a week apart to six healthy men. Rapid increase in LH, with peak concentrations was seen by 45 min post injection in all volunteers. There was a clear dose-dependent increase in LH concentrations in response to kisspeptin- 10 (P <0.0001). Area-Under-Curve analysis over 60 min following kisspeptin-10 administration showed 0.3 and 1μg/kg doses to be maximally stimulatory (P <0.01) with a reduced response at 3 μg/kg. Assessing the effect of steroid milieu: Question: Steroid feedback is central to the regulation of LH secretion: what effect does the steroid milieu have on LH responses to kisspeptin-10? Findings: The response to iv kisspeptin-10 (0.3μg/kg,) in the normal follicular phase (n=10) was compared with that in the presence of low endogenous sex steroids/high LH secretion (6 postmenopausal women) and in women taking combined contraceptive therapy (n=8) with suppressed LH secretion. Despite widely varying baseline secretion, LH increased significantly following kisspeptin-10 administration in the follicular phase (6.3±1.2 to 9.4±1.3 IU/L P=0.006), postmenopausal (35.3±2.8 to 44.7±3.4 IU/L P=0.005), etonogestrel (4.6±0.2 to 7.5±0.9 IU/L, P=0.02), and COCP groups (2.2±0.9 to 3.7±1.4 IU/L P<0.001). Pulse frequency study: Question: GnRH and LH secretion are pulsatile: can kisspeptin-10 enhance LH pulsatility? Findings: Four healthy men attended our clinical research facility for two visits five days apart for 10-min blood sampling. At the first visit, baseline LH pulsatility was assessed over a 9-hour period. During the second visit, an infusion of kisspeptin-10 was administered for 9 hours at 1.5μg/kg/hr after an hour of baseline sampling. LH pulse frequency increased in all subjects, with a mean increase from 0.7±0.1 to 1.0±0.2 pulses/hr (P = 0.01), with resultant increase in mean LH from 5.2±0.8 IU/L at baseline to 14.1±1.7 IU/L (P <0.01). High dose, longer duration infusion study: Question: Can kisspeptin-10 enhance testosterone secretion? Findings: Four healthy men attended our clinical research facility for a 34-hour supervised stay. Blood samples were collected at 10 min intervals for two 12 hour periods on consecutive days and hourly overnight. After 10.5 hours of baseline sampling a continuous intravenous infusion of kisspeptin-10 (4μg/kg/hr) was maintained for 22.5 hrs. Mean LH increased from 5.5±0.8 at baseline to 20.9±4.9 IU/L (P <0.05) and serum testosterone increased from 16.6±2.4 to 24.0±2.5 nmol/L (P <0.001). Translational studies in hypogonadal men with type 2 diabetes Question: Can kisspeptin-10 normalise testosterone secretion in hypogonadal men? Findings: Five hypogonadal men with T2DM (age 33.6±3 yrs, BMI 40.6±6.3, testosterone 8.5±1.0 nmol/L, LH 4.7±0.7 IU/L, HbA1c <8 %, duration of diabetes <5 yrs) and seven age matched healthy men were studied. Kisspeptin-10 was administered intravenous (0.3 μg/kg) with frequent (10-min) blood sampling. Mean LH increased in controls (5.5±0.8 to 13.9±1.7 IU/L P <0.001) and in T2DM (4.7±0.7 to 10.7±1.2 IU/L P=0.02) with comparable ΔLH (P=0.18). Baseline serum sampling for LH at 10-min intervals and hourly testosterone measurements were performed subsequently in four T2DM men for 12 hours. An intravenous infusion of kisspeptin-10 (4 μg/kg/hr) was administered 5 days later for 11 hours, with increases in serum LH (3.9±0.1 IU/L to 20.7±1.1 IU/L (P=0.03,) and testosterone (8.5±1.0 to 11.4±0.9 nmol/L, P=0.002). LH pulse frequency at baseline was lower in hypogonadal men with diabetes (0.6±0.1 vs. 0.8±0.1 pulses/hr, P=0.03) and increased to 0.9±0 pulses/hr (P=0.05). Translational studies in pubertal delay: Question: Defective Neurokinin B activity is associated with pubertal delay and the hierarchical interactions between kisspeptins and Neurokinin B remain to be elucidated: can kisspeptin-10 stimulate LH secretion with impaired Neurokinin B signalling? Findings: Four patients with TAC3 or TACR3 inactivating mutations presenting with delayed puberty were admitted for two 12 hr blocks of blood sampling every 10 min with vehicle (saline) or kisspeptin-10 (1.5 μg/kg/hour) infused intravenously. Mean LH and LH pulses frequency increased with kisspeptin-10 (P<0.05). However, four patients with Kallmann syndrome (with defective GnRH neuron migration), studied in parallel, did not respond, suggesting a potential diagnostic application for kisspeptin-10 in pubertal dysfunction. Conclusions In first-in-man studies of kisspeptin-10, it was demonstrated that endogenous LH pulse frequency can be enhanced in healthy men. The therapeutic potential of this finding in common reproductive endocrine disorders associated with decreased LH pulse frequency, i.e., late-onset male hypogonadism and pubertal dysfunction, was suggested in subsequent studies. Furthermore, kisspeptin signalling occurs upstream of GnRH neurons and is independent of Neurokinin B signalling in the central regulation of the hypothalamic-pituitary-gonadal axis.

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

The purpose of this study is to examine if testosterone shows reactivity to skydiving and to examine whether the testosterone level and reactivity was associated with sex and sensation seeking trait of the participants. Testosterone is an important steroid hormone which has several biological and socio-behavioral effects on people and is also present in disproportionate amounts in males and females; thus, it is important to explore how this hormone acts in different sex. Furthermore, exploring the relationship between sensation-seeking and testosterone could provide insight into the relation between psychological factor and hormonal response in humans. Forty-four people were recruited to participate in the study. The sample comprised of 73% males (N=32) and 27% females (N=12) with a mean age of 24 years (SD = 4.6) and an age range of 18 to 49. The participants volunteered to jump out of an airplane and give saliva samples at different time points during that day and during another day (basal levels). This study found that testosterone shows reactivity in response to skydiving, where the peak levels in males were higher than in females. It also found that people who scored higher in experience-seeking scores had higher testosterone level at jump than people who scored lower. Furthermore, it also revealed that people who scored higher in intension-seeking scores showed more reactivity in terms of testosterone i.e. the rise was steeper in these people. In summary, we see that psychological factors and sex predicted reactivity and peak level of testosterone after skydiving.

The Hypothalamic-Pituitary-Adrenal (HPA) and –Gonadal (HPG) axes have been considered mutually inhibitory; however, emerging evidence supports the proposition that this might not necessarily be the case. This idea is termed “coupling,” in which the HPA-HPG axis are mutually activated or deactivated. Coupling is examined across three data sets with different time-courses of stress exposure, and results demonstrate HPA-HPG co-activation occurs. Furthermore, stress exposure influences this relationship. The discussion shows how it is physiologically possible to have positive coupling or co-activation between these axes according to complex regulatory feedback systems and overlapping neural structures. Findings are interpreted developmentally, because adolescence may be a critical time for this co-activation to occur. Finally, the discussion emphasizes an individual difference perspective because each individual differs in the duration and type of stress they experience, and these exerted individualized effects on HPA-HPG coupling.

Introduction: The risk factors involved in treating transgender individuals with hormone therapy have been documented, but a full understanding of them remains elusive. Much of the research performed in transgender medicine is old or being completed overseas. It is thus the responsibility of current providers and investigators to expand our current knowledge, so this often marginalized population can receive the best quality care. Methods: A group of 28 transgender men and women who received care from the Endocrinology Clinic at Boston Medical Center were randomly selected as part of a quality control project to evaluate the risk factors involved in hormone therapy. Analysis was two-fold. First, change in lab values associated with known risk factors over a two-year period were assessed in individual patients. Second, group analysis sought to correlate changing hormone levels with lab values associated with known risk factors. Results: The result of the analysis was the majority of the patients who were observed did not suffer from any of the risk factors commonly associated with hormone therapy, and if anything benefited from the consistent clinical care. Several correlations were calculated between hormone levels and the lab values associated with the risk factors, however further analysis must be completed to confirm any connection. Discussion: The goal of this project was to not only evaluate care at Boston Medical Center, but also to draw attention to transgender health. In doing so, we have given an example of safe and effective hormone therapy and shown additional avenues for future research.

Glicocorticóides modulam a resposta imune de forma complexa em vertebrados expostos a diferentes estressores. Dado que as populações naturais têm estado expostas a uma multiplicidade de estressores, uma melhor compreensão da associação funcional entre a duração e a intensidade da resposta ao estresse, as mudanças resultantes nos níveis dos hormônios esteróides e seu impacto sobre os diferentes aspectos da imunocompetência emergem como um ponto chave para as estratégias de conservação dos vertebrados. Nós investigamos as relações entre os níveis plasmáticos de hormônios esteróides e a imunocompetência inata em anfíbios anuros, incorporando as metodologias de desafio de contenção, elevação experimental dos níveis de corticosterona por aplicação transdérmica, capacidade bactericida por espectrofotometria e o desafio imunológico com fitohemaglutinina. Nossos resultados demonstram que a capacidade bactericida plasmática (CBP) medida por espectrofotometria é um método confiável e preciso para estimar a imunocompetência de anfíbios anuros, além disso, mostramos a existência de uma grande diversidade interespecífica na CBP de anuros machos. Quando quatro diferentes espécies de Bufonídeos foram submetidas a um desafio de contenção, as respostas gerais incluíram aumento dos níveis plasmáticos de corticosterona (CORT) e da relação neutrófilo/linfócito (N:L) e diminuição dos níveis plasmáticos de testosterona (T). As respostas da CBP à contenção foram muito mais variáveis, com R. ictérica mostrando diminuição e R. marina mostrando aumento dos valores de CBP. Adicionalmente, CORT e N:L tenderam a aumentar mais em resposta à contenção com restrição de movimento do que à contenção sem restrição de movimento, indicando que os sapos demostraram um aumento da resposta ao estresse quando submetidos ao estressor mais intenso. Todas as variáveis estudadas mostraram variação interespecífica. Rhinella ornata apresentou os maiores níveis basais de CORT quando comparado com as outras espécies, enquanto R. ornata e R. ictérica mostraram os maiores valores basais de CBP. Entretanto, as mudanças na relação N:L, nos níveis de T e na CBP, não foram correlacionadas com o aumento em CORT, dentro ou entre espécies. A aplicação transdérmica de corticosterona eficientemente simulou eventos repetidos de resposta ao estresse agudo em Rhinella ictérica, sem alterar os parâmetros imunitários, mesmo após treze dias de tratamento. Curiosamente, o cativeiro a longo prazo não atenuou a resposta ao estresse, uma vez que estes sapos mantiveram um aumento de três vezes em CORT mesmo depois de três meses sob estas mesmas condições. Além disso, a manutenção em cativeiro a longo prazo, nas mesmas condições, aumentou a contagem total de leucócitos (TLC) e gerou uma diminuição ainda maior na CBP, sugerindo que as consequências da resposta ao estresse podem ser agravadas pelo tempo em cativeiro. Com base em nossos resultados, consideramos que uma avaliação cuidadosa é necessária para compreender a modulação da resposta imunitária pelo estresse a nível intra e interespecífico. A inclusão de diferentes segmentos da resposta imune é desejável, e a padronização da coleta de dados para todas as espécies sob o mesmo período (em geral, dentro ou fora da época reprodutiva) e mesma atividade (em geral, vocalizando ou forrageando) se faz obrigatória / Glucocorticoids modulate the immune response in complex ways in vertebrates exposed to different stressors. Given that natural populations have been exposed to a multitude of stressors, a better understanding of the functional association between duration and intensity of the stress response, the resulting changes in steroid hormone levels and their impact on different aspects of immunocompetence emerges as a cornerstone for vertebrate conservation strategies. We investigated the relationships between steroids levels and innate immunocompetence in anuran amphibians, incorporating the methodology of restraint challenge, experimental elevation of corticosterone levels by transdermal application, bacterial killing ability by spectrophotometry and the immune challenge with phytohemagglutinin. Our results demonstrate that the bacterial killing ability (BKA) measured by spectrophotometry is a reliable and accurate method to estimate the immunocompetence of anuran amphibians, additionally showed the existence of a large interspecific diversity in BKA from male anurans. When four different species of Bufonids were submitted to a restraint challenge, the general responses included increased in corticosterone plasma levels (CORT) and neutrophil/lymphocyte ratio (N:L) and decreased in testosterone plasma levels (T). The responses of BKA to restraint were much more variable, with R. icterica showing decreased and R. marina showing increased values. Additionally, CORT and N:L tended to increase more in response to restraint with movement restriction than to restraint without movement restriction, indicating that toads showed an increased stress response to the more intense stressor. All variables studied show interspecific variation. Rhinella ornate showed higher baseline CORT when compared to other species, while R. ornate and R. icterica showed the highest baseline BKA values. However, changes in N:L ratio, T levels and BKA, were not correlated to increased CORT within or between species. Transdermal application of corticosterone efficiently mimics repeated acute stress response events in Rhinella icterica, without changing the immune parameters even after thirteen days of treatment. Interestingly, long-term captivity did not mitigate the stress response, since the toads maintained three fold increased CORT even after three months under these conditions. Moreover, long-term captivity in the same condition increased total leukocyte count (TLC) and generated an even greater decrease in BKA, suggesting that consequences of the stress response can be aggravated by time in captivity. Based on our results, we consider that a careful evaluation is necessary in order to understand the modulation of the immune response by stress at intra and interspecific levels. The inclusion of different segments of the immune response is desirable, and a standardized data collection for all the species under the same period (e.g. inside or outside of breeding season) and same activity (e.g. calling or foraging) is mandatory