Orientador: José Mauricio Barbanti Duarte / Banca: Lindsay Unno Gimenes / Banca: Eveline dos Santos Zanetti / Banca: Denise Calisto Bongalhardo / Banca: Ricardo José Garcia Pereira / Resumo: O Brasil é o país com a maior diversidade de psitacídeos do mundo, abrigando 72 espécies reconhecidas, com 16 espécies presentes no "Livro Vermelho da Fauna Brasileira Ameaçada de Extinção". O papagaio-verdadeiro (Amazona aestiva) se destaca por sua popularidade como animal de estimação, sendo coletado na natureza em grande número para atender ao mercado ilegal de animais silvestres. Sua reprodução em cativeiro pode se tornar uma ferramenta para a conservação das populações na natureza, porém isso exige uma criação baseada em conhecimentos científicos e técnicas avançadas de reprodução. Esta tese teve como objetivos apresentar o perfil anual endócrino dos esteroides sexuais do Amazona aestiva e realizar um ensaio de estímulo hormonal através da aplicação de análogo de GnRH de liberação lenta na espécie. Utilizamos 10 casais e 4 machos adultos da espécie Amazona aestiva mantidos em viveiros suspensos, pertencentes ao Criadouro da Brisa, situado Jaboticabal/SP. As excretas foram coletadas ao menos uma vez por semana entre junho de 2011 e julho de 2012 para entendimento dos processos endócrinos que regem a reprodução da espécie e entre agosto de 2012 e dezembro de 2012 no ensaio de estímulo hormonal. O monitoramento da atividade gonadal foi feita de forma não invasiva por mensuração de metabólitos de andrógenos nas excretas dos machos e de progestágenos nas excretas de fêmeas. Foram coletadas amostras frescas de excretas, sempre no período entre 14h as 17h, e mantidas congeladas até o processamento. As amostras foram secas em estufa a 57oC, trituradas e os hormônios extraídos utilizando metanol a 80%. A dosagem hormonal foi realizada no Laboratório de Endocrinologia do NUPECCE (Núcleo de Pesquisa e Conservação de Cervídeos) utilizando ensaio imunoenzimático com o anticorpo para andrógenos e progestágenos. No ensaio de estimulo hormonal exógeno com analago de GnRH foi aplicado ... / Abstract: Brazil is the country with the greatest diversity of parrots in the world , with to 72 recognized species , with 16 species in the " Red List of Endangered Brazilian Wild Animals " . The blue-fronted amazon parrot ( Amazona aestiva ) stands out for its popularity as a pet , being collected from the wild in large numbers to attend the illegal market for wildlife. His captive breeding can become a tool for the conservation of populations in nature , but this requires a creation based on scientific knowledge and advanced breeding techniques . This thesis aimed to present the annual endocrine profile of sex steroids of Amazona aestiva and a test of hormonal stimulation by applying GnRH analogue of the slow release. It was used 10 couples and 4 adult males of Amazona aestiva kept in suspended cages, properties of the commercial breeder "Criadouro da Brisa", located in Jaboticabal / SP. The droppings were collected at least once a week between June 2011 and July 2012 for the understanding of endocrine processes of the reproduction in this specie and between August 2012 and December 2012 for testing hormonal stimulation . The monitoring of gonadal activity was noninvasively by measuring androgen metabolites in droppings of males and females of droppings progestogens. Fresh droppings samples were collected , always in between 14h to 17h , and kept frozen until processing . The samples were dried at 57oC, crushed and hormones were extracted using 80% methanol . The hormone dosage was performed at the Laboratory of Endocrinology, NUPECCE ( Center for Research and Conservation of Deer ) using enzyme immunoassay with antibody to androgens and progestins . In exogenous hormone stimulation test with GnRH was applied analago buserelin slow release in 5 couples and 5 couples were used to control the following excreta collection , processing and hormonal dosage previously described . The results of the annual listing of androgens in males ... / Doutor

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

No description available.

With approximately 8.5 million unwanted cats euthanized in the U.S. annually, convenient, cost effective methods of sterilization are greatly needed. Current spay/neuter techniques, such as surgery and hormonal intervention, are not satisfying this need due to their high cost, significant expertise required, and the need for feral cats to be collected and brought into clinics for treatment. The aim of this research is to develop a safe contraceptive vaccine that could be delivered to the feral cat population in bait without compromising non-feline species. Feline Herpes Virus (FHV) is a feline specific virus. The USDA has approved the immunization of cats with an attenuated, non-pathogenic strain of FHV expressing foreign antigens. In our research, we have partially replaced Glycoprotein I of FHV to express a fusion protein of Flagellin (FliC), Enhanced Green Fluorescent Protein (EGFP), and Gonadotropin Releasing Hormone (GnRH). FliC has been shown to stimulate a heightened antibody response when antigens are expressed as fusion proteins with it. GnRH, a major reproductive hormone responsible for the development of testes and ovaries in felines, is the target of our vaccine vector. Expression of EGFP will allow tracking of the viral vector. The expression of the fusion protein (FliC-EGFP-GnRH) is expected to stimulate an antibody and cell mediated immune response directed towards feline GnRH, which will provide an immunocontraceptive effect specific to cats. / Master of Science

A kisspeptina, codificada pelo gene KISS1, é um neuropeptídeo crucial na regulação do início da puberdade. A kisspeptina estimula a secreção hipotalâmica do hormônio liberador de gonadotrofinas (GnRH) após se ligar ao seu receptor GPR54. Mutações inativadoras do GPR54 são atualmente consideradas como uma causa rara de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Recentemente, uma mutação ativadora no receptor GPR54 foi implicada na patogênese da puberdade precoce dependente de gonadotrofinas (PPDG). Com base nesses achados, levantamos a hipótese de que alterações no gene KISS1 poderiam contribuir para a patogênese de distúrbios puberais centrais. O objetivo do presente estudo foi investigar a presença de variantes no gene KISS1 em pacientes com PPDG e HHI. Sessenta e sete crianças brasileiras com PPDG (63 meninas e 4 meninos) e 61 pacientes com HHI (40 homens e 21 mulheres) foram selecionados, incluindo casos esporádicos e familiares em ambos os grupos. A população controle consistiu de 200 indivíduos com história de desenvolvimento puberal normal. A região promotora e os 3 exons do gene KISS1 foram amplificados e submetidos a sequenciamento automático. Duas novas variantes no gene KISS1, p.P74S e p.H90D, foram identificadas em duas crianças não relacionadas, portadoras de PPDG idiopática. Ambas as variantes estão localizadas na região amino-terminal da kisspeptina-54 e estavam ausentes em 400 alelos controles. A variante p.P74S foi identificada em heterozigose em um menino que desenvolveu puberdade com um ano de idade. Sua mãe e avó materna, que apresentavam história de desenvolvimento puberal normal, eram portadoras da mesma variante em heterozigose, sugerindo penetrância incompleta e/ou herança sexo-dependente. A variante p.H90D foi identificada em homozigose em uma menina com PPDG, que desenvolveu puberdade aos seis anos de idade. Sua mãe, com história de menarca aos dez anos de idade, era portadora da mesma variante em heterozigose. Células transfectadas estavelmente com GPR54 foram estimuladas com concentrações crescentes de kisspeptina-54 (kp-54) humana selvagem ou contendo as mutações (kp-54 H90D e kp-54 P74S) e o acúmulo de fosfato de inositol (IP) foi medido. Nos estudos in vitro, a kp-54 P74S apresentou uma capacidade de ativação do receptor GPR54 semelhante à kp-54 selvagem. A kp-54 p.H90D mostrou uma ativação da sinalização do receptor significativamente mais potente que a kp-54 selvagem, sugerindo que essa é uma mutação ativadora. No grupo de HHI, uma nova variante (c.588-589insT) foi identificada em heterozigose na região 3 não traduzida do gene KISS1 em um paciente do sexo masculino. O papel dessa variante no fenótipo de HHI permanece indeterminado. Em conclusão, duas mutações no gene KiSS1 foram descritas pela primeira vez em associação com PPDG. / Kisspeptin, encoded by the KISS1 gene, is an important regulator of puberty onset. After binding to its receptor GPR54, kisspeptin stimulates gonadotropin-releasing hormone secretion by the hypothalamic neurons. Inactivating GPR54 mutations are a rare cause of normosmic isolated hypogonadotropic hypogonadism (IHH). Recently, a unique GPR54 activating mutation was implicated in the pathogenesis of gonadotropin dependent precocious puberty (GDPP). Based on these observations, we hypothesized that mutations in the KISS1 gene might be associated with central pubertal disorders. The aim of this study was to investigate KISS1 mutations in idiopathic GDPP and normosmic IHH. Sixty-seven Brazilian children (63 girls and 4 boys) with idiopathic GDPP and 61 patients with normosmic IHH (40 men and 21 women) were selected. Familial and sporadic cases were included in both groups. The control population consisted of 200 individuals who had normal timing of puberty. The promoter region and the 3 exons of the KISS1 gene were amplified and automatically sequenced. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in two unrelated children with idiopathic GDPP. Both mutations were absent in 400 control alleles and are located in the amino-terminal region of kisspeptin-54. The p.P74S mutation was identified in the heterozygous state in a boy who developed puberty at 1 yr of age. His mother and maternal grandmother, who had normal pubertal development, were also heterozygous for the p.P74S mutation, suggesting incomplete penetrance and/or sex-dependent inheritance. The p.H90D mutation was identified in the homozygous state in a girl with GDPP, who developed puberty at 6 yr of age. Her mother, who had menarche at 10 yr of age, carried the p.H90D mutation in the heterozygous state. CHO cells stably transfected with GPR54 were stimulated with different concentrations of synthetic human wild type or mutant kisspeptin-54 (KP54) and inositol phosphate (IP) accumulation was measured. In vitro studies revealed that the capacity of the p.P74S mutant KP54 to stimulate IP production was similar to the wild type. The p.H90D kisspeptin-54 showed a significantly more potent activation of GPR54 signaling in comparison to the wild type in vitro, suggesting a gain-of-function mutation. In the IHH group, a heterozygous variant in the 3 UTR of the KISS1 gene (c.588-589insT) was identified. The role of this variant in the IHH phenotype remains to be determined. In conclusion, two KiSS1 mutations were described for the first time in association with GDPP.

Objetivos: O objetivo deste estudo foi comparar os índices de Pulsatilidade (IP) e Resistência (IR) das artérias uterinas, o volume uterino e a espessura endometrial após o uso do Sistema Intra-uterino de Levonorgestrel (SIU-LNG) ou do agonista do GnRHa (GnRHa) em pacientes portadoras de endometriose pélvica. Pacientes e métodos: Setenta e nove mulheres voluntárias, com idade entre 18 e 40 anos, foram incluídas neste ensaio clínico comparativo, prospectivo, randomizado e controlado. Dezoito foram eliminadas do estudo baseadas nos critérios de exclusão, As 61 pacientes remanescentes foram divididas em dois grupos: 31 pacientes fizeram parte do grupo SIU-LNG (uma foi excluída antes da inserção por apresentar-se grávida) e 30 fizeram parte do grupo GnRHa. Foram submetidasa exame ultra-sonográfico transvaginal bidimensional no dia em que iniciaram o tratamento (inserção do SIU-LNG ou administração de uma ampola de 3,75 mg de GnRHa por via intra-muscular) e seis meses após, avaliando a espessura endometrial, o volume uterino e os IR e IP das artérias uterinas. Resultados: Ambos tratamentos promoveram redução da espessura endometrial (6.08±3.00mm para 2.70±0.98mm e 6.96±3.82mm para 3.23±2.32mm - média ±SD, grupo SIU-LNG e grupo GnRHa, respectivamente). O volume uterino teve redução no grupo usuário do GnRHa (86.67±28.38cm3 para 55.27±25.52cm3) sem alteração significativa nas usuárias do SIU-LNG (75.77±20.88cm3 para 75.97±26.62cm3). Em relação à ascularização uterina, notamos incremento dos IP das artérias uterinas em ambos os grupos (grupo SIU-LNG: artéria uterina direita de 2.38±0.72 para 2.76±0.99 (média ±SD) e artéria uterina esquerda 2.46±0.70 para 2.87±0.96, e grupo GnRHa: artéria uterina direita 2.04±0.59 para 3.12±0.98 eartéria uterina esquerda 2.24±0.59 para 3.15±0.89). Em relação ao IR das artérias uterinas, observamos incremento no grupo GnRHa em ambas artérias e somente na artéria uterina esquerda no grupo SIU-LNG (grupoSIU-LNG - artéria uterina direita de 0.85±0.08 para 0.88±0.07 e artéria uterina esquerda de 0.86±0.07 para 0.89±0.06, e grupo GnRHa: artéria uterina direita de 0.81±0.07 para 0.93±0.09 e artéria uterina esquerda 0.84±0.06 para 0.93±0.09). No entanto, ao compararmos as diferenças, a elevação foi significativamente maior nas usuárias do GnRHa. Conclusões: Ambos GnRHa e SIU-LNG promoveram redução na espessura endometrial e aumento no IP das artérias uterinas. Houve redução do volume uterino nas usuárias do grupo GnRHa, não se alterando no grupo SIU-LNG. Em relação ao IR, houve incremento em ambas as artérias nas usuárias de GnRHa e somente na artéria uterina esquerda nas usuárias do SIU-LNG. / Objectives:The objective of the present study was to compare the uterine arteries pulsatility index (PI) and resistence index (IR), uterine volume and endometrial thickness changes promoted by the use of the levonorgestrel intrauterine device (LNG-IUD) and the gonadotropin-releasing hormone analogue (GnRHa)in patients with endometriosis. Methods: Seventy nine women aged 18 to 40 years were included in this randomized controlled trial. Eighteen was excluded based on the exclusion criteria. The patients were randomly allocated in two groups: 31 women who used the LNG-IUD (since one became pregnant before insertion and wasexcluded) and 30 who used monthly GnRHa injections. They were submitted to a transvaginal two dimensional ultrasound scan on the day the treatment started and 6 months later, for the evaluation of uterine arteries PI, uterine arteries RI, uterine volume and endometrial thickness. Results: The use of LNG-IUD promoted an ndometrial thickness decrease (6.08±3.00mm to 2.7±0.98mm; mean±SD) as does the use of GnRHa (6.96±3.82mm to 3.23±2.32mm). The uterine volume decreased in the GnRHa group (86.67±28.38cm3to 55.27±25.52cm3), but not in the LNG-IUD group (75.77±20.88cm3 to 75.97±26.62cm3). Uterine arteries PI increased in both groups : Uterine arteries PI: LNG-IUD right uterine arterie 2.38 ± 0.72 to 2.76 ± 0.99 and left uterine arterie 2.46 ± 0.70 to 2.87 ± 0.96, and GnRHa right uterine arterie 2.04 ± 0.59 to 3.12 ± 0.98 and left uterine arterie 2.24±0.59 to 3.15 ± 0.89. Uterine arteries RI increased in both arteries in GnRHa and only in the left uterine arterie in the LNG-IUD :Uterine arteries RI : LNG-IUD right uterine arterie 0.85 ± 0..08 to 0.88 ± 0.07 and left uterine arterie 0.86 ± 0.07 to 0.89 ± 0.06, and GnRHa right uterine arterie 0.81 ± 0.07 to 0.93 ± 0.09 and left uterine arterie 0.84 ± 0.06 to 0.93 ± 0.09 . However, the increase was significant higher in the GnRHa group. Conclusions: Both GnRHa and LNG-IUD promoted an endometrial thickness decrease and an increase in the uterine arteries PI. The uterine volume decreased in women who used GnRHa, but not in those who used LNG-IUD.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

Ιστορικά, τα ευρήματα των C.B. Huggins (Huggins and Hodges, 1941; Huggins, 1963) και A.V. Schally (Schally et al., 1984) αποτέλεσαν την απαρχή μιας ερευνητικής πορείας με αφετηρία το πεδίο της νευροενδοκρινολογίας και προορισμό εκείνα της γυναικολογίας και της ογκολογίας. Η γνώση αναφορικά με τη φυσιολογία της ενδογενούς ορμόνης (LHRH) και το ρόλο της στην παθοβιοχημεία των ασθενειών (ενδοκρινικών διαταραχών και ορμονο – εξαρτώμενων καρκίνων) και υπό το πρίσμα της πρωτεύουσας ή υποστηρικτικής θεραπευτικής προσέγγισης, κατέστησε τον ιατρικό ευνουχισμό μέσω της δράσης στον υποδοχέα της LHRH – Ι στρατηγική επιλογής για την καταπολέμηση των ενδοκρινικών διαταραχών και των ορμονο – εξαρτώμενων καρκίνων (καρκίνος του μαστού, καρκίνος των ωοθηκών, καρκίνος του ενδομητρίου, καρκίνος του προστάτη). Μια πληθώρα αναλόγων της LHRH έδωσε και δίνει το παρόν σε προκλινικό και κλινικό επίπεδο, με τη συστηματική έρευνα, σύνθεση και ανάπτυξη να αποδίδουν μόρια – αγωνιστές ή – ανταγωνιστές του υποδοχέα της LHRH – Ι, πεπτιδικής (γραμμική ή κυκλική δομή, stapled peptides) ή μη φύσης ως μοναδιαίες οντότητες ή σε σύζευξη με ένα ευρύ φάσμα κυτταροτοξικών μορίων (Αnderes et al., 2003; Keramida et al., 2006; Persson et al., 2009; Kritzer, 2010; Mezo and Manea, 2010). Η παρούσα διδακτορική διατριβή αποσκοπεί στην in vitro και in vivo αξιολόγηση και φαρμακοκινητική μελέτη καινοτόμων αναλόγων της LHRH – κυκλικής και γραμμικής δομής – στη βάση του ορθολογικού μοριακού σχεδιασμού και αποσκοπώντας σε εναλλακτικές προσεγγίσεις για την καταπολέμηση του καρκίνου του προστάτη (και έτερων ενδοκρινικών διαταραχών), συγκριτικά με το leuprolide (εμπορικά διαθέσιμος αγωνιστής του υποδοχέα της LHRH – Ι). Η παρούσα διδακτορική διατριβή θέτει ως υπόθεση εργασίας πως καινοτόμα ανάλογα της LHRH των ήδη υπάρχοντων στην κλινική με βελτιωμένο φαρμακοκινητικό προφίλ δύναται να αποτελέσουν τη βάση βέλτιστων εναλλακτικών θεραπευτικών προσεγγίσεων με ενισχυμένη αποτελεσματικότητα και μειωμένη τοξικότητα, δρώντας in situ ή/ και προσφέροντας νέες δυνατότητες χορήγησης, εστιάζοντας στην οδό ή/ και τη μείωση της συχνότητας αυτής. Η εν λόγω ερευνητική προσέγγιση εστιάζει στον καρκίνο του προστάτη, μια νόσο που χρήζει εναλλακτικής θεραπευτικής στρατηγικής, δεδομένης της χαμηλής αποτελεσματικότητας αυτής (η νόσος προοδευτικά γίνεται ανεξάρτητη των ορμονών και συνεπώς, μεταστατική), αλλά και της χαμηλής ποιότητας ζωής των ασθενών στη βάση του «συμβιβασμού» με τις οδούς χορήγησης που εφαρμόζονται σήμερα στην κλινική (depot formulations). Επιπρόσθετα και κατά την εκπόνηση της εν λόγω ερευνητικής προσέγγισης αναπτύχθηκαν, επικυρώθηκαν και βελτιστοποιήθηκαν καινοτόμες μεθοδολογίες υγρής χρωματογραφίας – φασματομετρίας μάζας (LC – MS/ MS) σε βιολογικά υγρά – ηπατικά μικροσώματα (μύα, επίμυα, ανθρώπου), νεφρικές μεμβράνες μύα, πλάσμα και όρχεις μύα – και έτερα υποστρώματα (κυτταρικά εκχυλίσματα, υδάτινο περιβάλλον ιχθύων Danio rerio), οι οποίες, εν συνεχεία, συζεύχθηκαν με in vitro ή/ και in vivo βιοδοκιμασίες. Τα πειραματικά ευρήματα συγκρίθηκαν με εκείνα των εμπορικά διαθέσιμων αναλόγων της LHRH, με αγωνιστική (leuprolide) ή ανταγωνιστική δράση (antide, cetrorelix). Πιο αναλυτικά και λαμβάνοντας υπόψην το «νοσηρό» φαρμακοκινητικό προφίλ των αναλόγων της LHRH που χρησιμοποιούνται σήμερα στην κλινική, διερευνήθηκε η in vitro (ηπατικά μικροσώματα μύα, επίμυα και ανθρώπου – νεφρικές μεμβράνες μύα) και in vivo (πλάσμα μύα) πεπτιδική σταθερότητα των υπό μελέτη αναλόγων, συγκριτικά με την LHRH και εμπορικά διαθέσιμα ανάλογα αυτής (antide, leuprolide). Τα ευρήματα επέτρεψαν την αξιολόγηση και ταξινόμηση των αναλόγων του ενδιαφέροντος βάσει πεπτιδικής σταθερότητας (δοκιμασία σάρωσης). Ταυτόχρονα, προσδιορίστηκε το μεταβολικό τους προφίλ, αποκαλύπτοντας τους ευάλωτους πεπτιδικούς δεσμούς, καθώς και τις εμπλεκόμενες ενδοπεπτιδάσες (NEP – EC 3.4.24.11, ACE – EC 3.4.15.1). Τα ευρήματα επιβεβαιώθηκαν περαιτέρω με τη χρήση ειδικών ενζυμικών αναστολέων (ενδεικτικά: DL – Thiorphan). Ο νεφρός βρέθηκε να είναι το πρωτεύον μεταβολικό όργανο. Η ανίχνευση και η ημι – ποσοτικοποίηση των μεταβολικών προϊόντων οδήγησε στον προσδιορισμό σχέσεων δομής – δραστικότητας, αποτελώντας τη βάση του ορθολογικού σχεδιασμού καινοτόμων μορίων. Οι απορρέουσες σχέσεις δομής –δραστικότητας υποστηρίζουν πως (i) η μεθυλίωση της υδροξυλομάδας της Tyr5, (ii) η αντικατάσταση της Pro9 από Aze και (iii) η κυκλοποίηση ενισχύουν την πεπτιδική σταθερότητα των αναλόγων του ενδιαφέροντος. Στα πλαίσια μελετών φαρμακοδυναμικής, αναπτύχθηκε καινοτόμος μεθοδολογία LC – MS/ MS για τον ταυτόχρονο ποσοτικό προσδιορισμό της τεστοστερόνης και των υπό μελέτη αναλόγων σε πλάσμα (0, 05 – 100 ng/mL) και όρχεις μύα (2, 0 – 2000 ng/g). Η τεστοστερόνη θεσπίστηκε βιοδείκτης αποτελεσματικότητας (efficacy) και τοξικότητας (toxicity) στη βάση του καίριου ρόλου που διαδραματίζει το εν λόγω στεροειδές στη φυσιολογία του άξονα υποθάλαμος – υπόφυση – γονάδες και την παθοβιοχημεία των ενδοκρινικών διαταραχών και του ορμονο – εξαρτώμενου καρκίνου του προστάτη. Η φαρμακολογική απόκριση (απελευθέρωση τεστοστερόνης) βρέθηκε πως είναι ειδική και λαμβάνει χώρα μέσω του υποδοχέα της LHRH – Ι. Πιο σημαντικά, επετεύχθη ιατρικός ευνουχισμός, βάσει ιστοπαθολογικών ευρημάτων και προσδιορισμού της τεστοστερόνης (πλάσμα και όρχεις μύα), κατόπιν επαναλαμβανόμενης ενδοπεριτοναϊκής χορήγησης του επιλεγμένου καινοτόμου γραμμικού αναλόγου της LHRH, linearGnRH1. Το ίδιο ανάλογο βρέθηκε να έχει κυτταροστατική δράση σε πειράματα κυτταρικού πολλαπλασιασμού (κύτταρα LNCaP και PC3). Παράλληλα και υπό το πρίσμα του ρόλου της LHRH στην παθοβιοχημεία έτερων ορμονο – εξαρτώμενων καρκίνων (καρκίνος του μαστού, καρκίνος των ωοθηκών, καρκίνος του ενδομητρίου), αναπτύχθηκε, επικυρώθηκε και βελτιστοποιήθηκε καινοτόμος μεθοδολογία LC – MS/ MS για τον ποσοτικό προσδιορισμό της 17β – οιστραδιόλης σε βιολογικά υγρά. Τέλος, αναπτύχθηκε καινοτόμος μεθοδολογία LC – MS/MS για τον ποσοτικό προσδιορισμό πεπτιδίων του ενδιαφέροντος στο υδάτινο περιβάλλον των ιχθύων του είδους Danio rerio. Το καινοτόμο γραμμικό ανάλογο της LHRH, linearGnRH1, δεδομένου του φαρμακοκινητικού/ φαρμακοδυναμικού του προφίλ, δρα υποστηρικτικά ως προς την υπόθεση της παρούσας διδακτορικής διατριβής, σύμφωνα με την οποία καινοτόμα ανάλογα των ήδη υπάρχοντων στην κλινική με βελτιωμένο φαρμακοκινητικό προφίλ δύναται να αποτελέσουν τη βάση βέλτιστων εναλλακτικών θεραπευτικών προσεγγίσεων με ενισχυμένη αποτελεσματικότητα και μειωμένη τοξικότητα, δρώντας in situ ή/ και προσφέροντας νέες δυνατότητες χορήγησης, εστιάζοντας στην οδό ή/ και τη μείωση της συχνότητας αυτής. Το linearGnRH1 δύναται να αποτελέσει τη βάση για τον ορθολογικό σχεδιασμό μορίων (stapled peptides, μιμητές), αποσκοπώντας σε εναλλακτικές θεραπευτικές στρατηγικές για την καταπολέμηση του ορμονο – εξαρτώμενου καρκίνου ή/ και των ενδοκρινικών διαταραχών. Πέραν του linearGnRH1, ας σημειωθεί πως κατά την εκπόνηση της παρούσας διδακτορικής διατριβής συγκεκριμένα καινοτόμα κυκλικά ανάλογα της LHRH (cyclicGnRH1, cyclicGnRH2, cyclicGnRHDL1, cyclicGnRHDL2) εμφάνισαν ένα διακριτό φαρμακοκινητικό προφίλ. Το φαρμακοδυναμικό προφίλ των εν λόγω κυκλικών πεπτιδίων απαιτείται να διερευνηθεί περαιτέρω με την εφαρμογή των καινοτόμων μεθοδολογιών LC – MS/MS που αναπτύχθηκαν, επικυρώθηκαν και βελτιστοποιήθηκαν κατά την εκπόνηση της παρούσας διδακτορικής διατριβής. Συνολικά, η καινοτομία της εν λόγω ερευνητικής προσέγγισης έγκειται (i) στην ανάπτυξη, επικύρωση και βελτιστοποίηση ενός πλήθους μεθοδολογιών LC – MS/MS σε σύζευξη με in vitro και in vivo βιοδοκιμασίες, οι οποίες και αποτελούν ένα διακριτό αναλυτικό εργαλείο και (ii) στο προκλινικό φαρμακολογικό μοντέλο που αναπτύχθηκε με την επιλογή του μύα ως ζωικό πρότυπο. Κοινή συνισταμένη, η εν τω βάθει αξιολόγηση της φαρμακοκινητικής/ φαρμακοδυναμικής των πεπτιδίων του ενδιαφέροντος με το ερευνητικό βλέμμα στα πεπτιδικά φάρμακα.. stapled peptides.. μιμητές.. / The highly influential findings of C. B. Huggins (Huggins, 1963) and A. V. Schally (Schally et al., 1984) brought a new era in the research fields of neuroendocrinology, gynecology and oncology. The knowledge acquired regarding the physiology of LHRH and its role in the pathobiochemistry of the disease resulted in the consideration of medical castration via the LHRH receptor as a well established strategy for the treatment of endocrine disorders (e.g. precocious puberty) and hormone – dependent cancers (breast cancer, endometrial cancer, prostate cancer). Numerous LHRH analogues have been synthesized and evaluated both in the clinic and preclinical level. Overall, systematic work has resulted in the synthesis of LHRH receptor agonists and antagonists, either peptides (linear, cyclic, stapled peptides) or small organic molecules as entities or combined with various cytotoxic molecules (Αnderes et al., 2003; Keramida et al., 2006; Persson et al., 2009; Kritzer, 2010; Mezo and Manea, 2010). Although extensive research has been carried out in the field of hormonal therapy, poor pharmacokinetic properties still characterize LHRH peptide analogues. Poor stability of LHRH analogues compromises efficacy, while the need for their subcutaneous administration (depot formulations) aggravates the quality of life for cancer patients. Nowadays, LHRH analogues in the clinic most likely achieve the desired pharmacologic effects by action primarily on the pituitary and to a much lesser extent by direct antiproliferative effects on tumor cells. Herein, we hypothesize that stable analogues of such super – agonists would be advantageous, either by allowing a reduction in dosing frequency or by allowing the use of analogues that act directly on the tumor, with possible additive effects and subsequent enhancements in efficacy. In this context, the pharmacokinetic/ pharmacodynamic profiles of novel LHRH analogues were determined both in vitro and in vivo. Similarly, commercially available LHRH analogues (leuprolide, antide, cetrorelix) served as positive controls. Novel LC – MS/MS based approaches (LC – MS/ MS methodologies coupled to in vitro and in vivo bioassays) were developed, validated and optimized for the evaluation of the peptide analogues in question (linear or cyclic) in various biological fluids and matrices; (i) mouse, rat and human liver microsomes, (ii) mouse kidney membrane preparations, (iii) mouse plasma and tissue (testis), (iv) egg – water (Danio rerio embryos environment). Furthermore, a novel LC – MS/MS based approach was developed, validated and optimized for the simultaneous quantification of testosterone and peptides in question, upon the intraperitoneal administration of peptide analogues in mice. Testosterone served as an efficacy and toxicity biomarker. Peptide metabolism was thoroughly studied by an LC – MS/MS based approach coupled to an in vitro bioassay (mouse kidney membrane preparations), allowing (i) structural elucidation and semi – quantitation of metabolites (and peptides) as a function of time, (ii) determination of the susceptible to proteolysis peptide – bonds, (iii) structure – activity relationships (SARs) and (iv) peptide ranking (screening assay). Taking into account the role of LHRH in gynecology and hormone – dependent cancers of breast, endometrium and ovary, an LC – MS/MS based approach was developed for the quantification of 17β – oestradiol (efficacy and toxicity biomarker) in mouse plasma upon the intraperitoneal administration of peptide analogues in mice. A novel LC – MS/MS based approach was also developed for the quantification of peptides of interest in the aquatic environment of Danio rerio. Employing the aforementioned analytical tools, peptide stability against proteolysis was evaluated both in vitro (mouse kidney membrane preparations) and in vivo (mouse plasma). LHRH and commercially available analogues served as positive controls. The SARs derived suggested that enhanced stability was achieved by (i) methylation on the hydroxyl group of Tyr5, (ii) replacement of Pro9 by Aze and (iii) cyclisation. Hence, new promising chemical entities could be synthesized and developed on the basis of rational drug design. Metabolic profiles were also determined revealing the susceptible to proteolysis peptide bonds. Susceptible peptide bonds and endopeptidases involved were further confirmed in the presence of specific endopeptidase inhibitors. A facile preclinical mouse model was developed in the context of our objectives. Intraperitoneal administration was selected, since (i) it is a mix – mode type of administration with elements of rapid absorption and (ii) oral administration was not practical due to the low bioavailability of the peptides that were tested. The LC – MS/MS based quantification of the selected bioactive peptides and their corresponding metabolites as well as the selective monitoring of biomarkers (e.g. testosterone) in response to drug dose, in plasma and testes, combined with the appropriate preclinical mouse model, represents a distinctive approach. The mouse model described in this paper is particularly valuable, since (i) the human LHRH receptor is homologous to the mouse receptor (Millar, 2004), (ii) information on in vitro and in vivo stability can be obtained with a relatively small amount of peptide (1 – 2 mg), (iii) information on the LHRH receptor agonism (receptor specific in vivo modulation) can be obtained by using testosterone as a marker, (iv) it allows the determination of the dosing regimen required for efficacy based on action on the pituitary, (v) it can become the basis of follow up experiments on genetically modified mouse animal models or other tumour xenografted mouse models (Sharpless and Depinho, 2006; Morgan et al., 2008). The robust sensitive methodology that was developed for the quantification of testosterone in mouse plasma (0.05 – 100 ng/mL) or determination of testosterone in testes (2 – 2000 ng/g) provides an excellent handle on compound efficacy assessment. Testosterone as an efficacy and toxicity biomarker was found to be specific upon peptide binding to the LHRH receptor. Medical castration was achieved upon the repeated dosing of a selected novel linear analogue (linearGnRH1). Measurements in plasma were further supported by statistical significant testosterone values in testis and histopathological findings (atrophy). Moreover the testis weights of the treated animals were significantly lower in comparison to the control group (atrophy induced by dosing), thus making the differences in testosterone testis concentration between control and peptide treated animals even more pronounced. Although the binding affinity of linearGnRH1 on the LHRH receptor was not as high as the binding affinity of leuprolide (~ 15 nM versus <1 nM), the in vivo efficacy between the two analogues was similar (at the tested dose), suggesting that the enhanced stability or bioavailability of linearGnRH1, compensates for binding affinity differences. LinearGnRH1 was also found to be anti – proliferative upon dosing in LNCaP cells (as potent as the superagonist leuprolide). It is possible that linearGnRH1 can play a significant role for the treatment of hormone – dependent cancers, by acting not only at the pituitary level (thus, suppressing the pituitary – testicular axis), but also by exerting an antitumor activity directly on cancer cells, as has been previously shown for other LHRH agonists (Maudsley et al., 2004; Marelli et al., 2006). Except for linearGnRH1, selected cyclic analogues (cyclicGnRH1, cyclicGnRH2, cyclicGnRHDL1, cyclicGnRHDL2) exhibited a distinct pharmacokinetic profile. Their pharmacodynamic profiles should be evaluated further employing the novel LC – MS/MS based approaches developed and validated in this study. Overall, the novelty of the approach described herein consists of (i) the LC – MS/MS methodologies coupled with in vitro and in vivo bioassays developed, validated and employed that provide a distinct analytical tool and (ii) the facile preclinical pharmacological mouse model developed and employed. The approach aims to the pharmacokinetic/pharmacodynamic evaluation of the peptides of interest towards a new generation of peptide drugs.. stapled peptides.. mimetics. Considering the pharmacokinetic/ pharmacodynamic profiles of linearGnRH1, findings on this novel analogue satisfy our hypothesis according to which stable analogues of the LHRH super – agonists used in the clinic would be advantageous, either by allowing a reduction in dosing frequency or by allowing the use of analogues that act directly on the tumor, with possible additive effects and subsequent enhancements in efficacy. LinearGnRH1 can serve as the platform for the rational drug design of new chemical entities (stapled peptides, mimetics) for the treatment of hormone – dependent cancers and/ or endocrine disorders.

A kisspeptina, codificada pelo gene KISS1, é um neuropeptídeo crucial na regulação do início da puberdade. A kisspeptina estimula a secreção hipotalâmica do hormônio liberador de gonadotrofinas (GnRH) após se ligar ao seu receptor GPR54. Mutações inativadoras do GPR54 são atualmente consideradas como uma causa rara de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Recentemente, uma mutação ativadora no receptor GPR54 foi implicada na patogênese da puberdade precoce dependente de gonadotrofinas (PPDG). Com base nesses achados, levantamos a hipótese de que alterações no gene KISS1 poderiam contribuir para a patogênese de distúrbios puberais centrais. O objetivo do presente estudo foi investigar a presença de variantes no gene KISS1 em pacientes com PPDG e HHI. Sessenta e sete crianças brasileiras com PPDG (63 meninas e 4 meninos) e 61 pacientes com HHI (40 homens e 21 mulheres) foram selecionados, incluindo casos esporádicos e familiares em ambos os grupos. A população controle consistiu de 200 indivíduos com história de desenvolvimento puberal normal. A região promotora e os 3 exons do gene KISS1 foram amplificados e submetidos a sequenciamento automático. Duas novas variantes no gene KISS1, p.P74S e p.H90D, foram identificadas em duas crianças não relacionadas, portadoras de PPDG idiopática. Ambas as variantes estão localizadas na região amino-terminal da kisspeptina-54 e estavam ausentes em 400 alelos controles. A variante p.P74S foi identificada em heterozigose em um menino que desenvolveu puberdade com um ano de idade. Sua mãe e avó materna, que apresentavam história de desenvolvimento puberal normal, eram portadoras da mesma variante em heterozigose, sugerindo penetrância incompleta e/ou herança sexo-dependente. A variante p.H90D foi identificada em homozigose em uma menina com PPDG, que desenvolveu puberdade aos seis anos de idade. Sua mãe, com história de menarca aos dez anos de idade, era portadora da mesma variante em heterozigose. Células transfectadas estavelmente com GPR54 foram estimuladas com concentrações crescentes de kisspeptina-54 (kp-54) humana selvagem ou contendo as mutações (kp-54 H90D e kp-54 P74S) e o acúmulo de fosfato de inositol (IP) foi medido. Nos estudos in vitro, a kp-54 P74S apresentou uma capacidade de ativação do receptor GPR54 semelhante à kp-54 selvagem. A kp-54 p.H90D mostrou uma ativação da sinalização do receptor significativamente mais potente que a kp-54 selvagem, sugerindo que essa é uma mutação ativadora. No grupo de HHI, uma nova variante (c.588-589insT) foi identificada em heterozigose na região 3 não traduzida do gene KISS1 em um paciente do sexo masculino. O papel dessa variante no fenótipo de HHI permanece indeterminado. Em conclusão, duas mutações no gene KiSS1 foram descritas pela primeira vez em associação com PPDG. / Kisspeptin, encoded by the KISS1 gene, is an important regulator of puberty onset. After binding to its receptor GPR54, kisspeptin stimulates gonadotropin-releasing hormone secretion by the hypothalamic neurons. Inactivating GPR54 mutations are a rare cause of normosmic isolated hypogonadotropic hypogonadism (IHH). Recently, a unique GPR54 activating mutation was implicated in the pathogenesis of gonadotropin dependent precocious puberty (GDPP). Based on these observations, we hypothesized that mutations in the KISS1 gene might be associated with central pubertal disorders. The aim of this study was to investigate KISS1 mutations in idiopathic GDPP and normosmic IHH. Sixty-seven Brazilian children (63 girls and 4 boys) with idiopathic GDPP and 61 patients with normosmic IHH (40 men and 21 women) were selected. Familial and sporadic cases were included in both groups. The control population consisted of 200 individuals who had normal timing of puberty. The promoter region and the 3 exons of the KISS1 gene were amplified and automatically sequenced. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in two unrelated children with idiopathic GDPP. Both mutations were absent in 400 control alleles and are located in the amino-terminal region of kisspeptin-54. The p.P74S mutation was identified in the heterozygous state in a boy who developed puberty at 1 yr of age. His mother and maternal grandmother, who had normal pubertal development, were also heterozygous for the p.P74S mutation, suggesting incomplete penetrance and/or sex-dependent inheritance. The p.H90D mutation was identified in the homozygous state in a girl with GDPP, who developed puberty at 6 yr of age. Her mother, who had menarche at 10 yr of age, carried the p.H90D mutation in the heterozygous state. CHO cells stably transfected with GPR54 were stimulated with different concentrations of synthetic human wild type or mutant kisspeptin-54 (KP54) and inositol phosphate (IP) accumulation was measured. In vitro studies revealed that the capacity of the p.P74S mutant KP54 to stimulate IP production was similar to the wild type. The p.H90D kisspeptin-54 showed a significantly more potent activation of GPR54 signaling in comparison to the wild type in vitro, suggesting a gain-of-function mutation. In the IHH group, a heterozygous variant in the 3 UTR of the KISS1 gene (c.588-589insT) was identified. The role of this variant in the IHH phenotype remains to be determined. In conclusion, two KiSS1 mutations were described for the first time in association with GDPP.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-12-19T18:38:39Z No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-22T11:01:46Z (GMT) No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-22T11:01:46Z (GMT). No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-07-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infertility affects more couples and assisted reproduction techniques offer a possibility of treatment and the chance of having a child. Thus, the first attempt to ovulation induction is critical to the success of the cycle or even for future attempts is successful. Objective: To compare the protocols using GnRH agonist or antagonist for ovarian stimulation in normo-responders undergoing the first cycle of IVF/ICSI. Methods: we conducted a literature review on the history of ovulation induction controlled by medications. From the data available in the database of electronic medical records SISFERT used in the Laboratory of Human Reproduction (LabRep-HC-FM-UFG) a comparative retrospective observational study was conducted with 50 patients divided into two groups according to protocol: GnRH-agonist (leuprolide acetate 1 mg/day short protocol) or GnRHantagonist (Cetrorelix 0.25 mg/day), which received 150 IU/day of rFSH (follitropin alpha) and 250 µg of rhCG (alpha-coriogonadotrofina) in both groups. Results: Statistically significant differences were observed in the days of stimulation with rFSH, total dose of gonadotropin, days of use of GnRH, GnRH dose and total number of follicles (≥ 16 mm) on the day of the group rhCG GnRH agonist. There was no significant difference in other parameters, however, the number of oocytes retrieved was slightly higher in the GnRH agonist, but fertilization rate was higher in the GnRH-antagonist. Pregnancy rates and clinical chemistry were similar in both groups. Conclusions: although no significant differences in the results analyzed, the use of flexible antagonist protocol facilitates the handling and enables the patient using much lower doses of gonadotropins itself as the antagonist, reducing the cost of treatment when compared to the protocol with GnRH agonist. / A infertilidade afeta cada vez mais casais e as técnicas de reprodução assistida oferecem uma possibilidade de tratamento e a chance de ter um filho. Assim, a primeira tentativa de indução da ovulação é fundamental para o sucesso do ciclo ou, até mesmo, para que tentativas futuras sejam bem sucedidas. Objetivo: comparar os protocolos utilizando agonista ou antagonista do GnRH para estimulação ovariana em pacientes normo-respondedoras submetidas ao primeiro ciclo de FIV/ICSI. Métodos: foi realizada uma revisão da literatura sobre a história da indução da ovulação controlada por medicamentos. A partir dos dados disponíveis no banco de prontuários eletrônicos SISFERT utilizado pelo Laboratório de Reprodução Humana (LabRep–HC–FM–UFG), um estudo observacional retrospectivo comparativo foi conduzido com 50 pacientes distribuídas em dois grupos de acordo com o protocolo: GnRH-agonista (acetato de leuprolide 1 mg/dia protocolo curto) ou GnRH-antagonista (cetrorelix 0,25 mg/dia); e que receberam 150 UI/dia de rFSH (alfa-folitropina) e 250 µg de rhCG (alfa-coriogonadotrofina) em ambos os grupos. Resultados: foram observadas diferenças estatisticamente significativas nos dias de estimulação com rFSH, dose total de gonadotrofina, dias de uso do GnRH, dose total de GnRH e o número de folículos (≥ 16 mm) no dia do rhCG no grupo GnRH-agonista. Não houve diferença significativa nos outros parâmetros, no entanto, o número de oócitos recuperados foi ligeiramente maior no grupo GnRH-agonista, mas a taxa de fertilização foi maior no grupo GnRH-antagonista. As taxas de gravidez química e clínica foram similares nos dois grupos. Conclusões: embora não tenha havido diferenças significativas nos resultados analisados, o uso do protocolo flexível com antagonista facilita a manipulação pela paciente usuária e possibilita doses menores tanto de gonadotrofinas quanto do próprio antagonista, reduzindo o custo do tratamento quando comparado ao protocolo com agonista do GnRH