Salamanders are one of the most at-risk taxa in the world due to habitat destruction, pollution, climate change, invasive diseases, and more. This has led to a need for conservation breeding programs that are often associated with zoos and aquaria. Salamanders can be difficult to breed in captivity, though, due to their dependence on specific environmental cues and other unknown factors that stimulate sperm and egg production. To overcome these challenges, assisted reproductive technologies (ART) such as exogenous hormone administration, sperm cryopreservation, and in-vitro fertilization have been developed to increase offspring propagation and maintain genetic diversity. If genetically robust populations of salamander species can be sustainably managed ex situ into the future, then their species can be protected in situ through practices such as reintroductions into native habitats. Given the importance of salamanders to healthy, functioning ecosystems, my doctoral research focused on methods to advance caudate conservation efforts through ART. This dissertation addresses four key areas targeting salamander reproduction for conservation: 1) Novel, non-invasive hormone administration routes; 2) Sperm extender toxicity; 3) Novel sperm cryoprotectants; and 4) Application of ART to target salamander species. The objectives were to: 1) Compare nasal, oral, and intramuscular delivery routes of gonadotropin-releasing hormone on spermic response; 2) Compare sperm extenders at varying osmolalities for maintaining sperm quality over time; 3) Test dimethyl sulfoxide versus dimethylformamide as cryoprotectants to increase frozen-thawed sperm viability, motility, and fertilization capability in the eastern tiger salamander (Ambystoma tigrinum); and 4) Transfer ART protocols developed from the eastern tiger salamander to the eastern hellbender (Cryptobranchus alleganiensis) and spotted salamander (Ambystoma maculatum). This work answers critical questions that should help advance salamander ART research into the future and lead to more sustainably managed caudate populations.

Infertilitet är ett relativt vanligt hälsoproblem i dagens samhälle. Infertilitet kan uppstå som resultat av en sjukdom där en störning av reproduktiva kroppsfunktioner hindrar uppkomsten av en graviditet och födsel av ett barn. Flertalet kvinnor som senarelägger starten på barnafödandet löper en större risk att drabbas av primär och sekundär infertilitet. Barnlöshet går inte alltid att förebygga och alla kvinnor som önskar att få barn kan inte alltid få barn. Syftet med det här arbetet var att belysa kvinnors upplevelse av ofrivillig barnlöshet efter misslyckade försök att bli mamma. Litteraturöversikt är den metod som har valts för att besvara syftet med det här arbetet. Integrerad analys är den dataanalysmetod som har används. Resultatet visar att isolering från omgivningen är vanlig förekommande bland kvinnor som är ofrivilligt barnlösa. Ofrivilligt barnlösa kvinnor kunde uppleva sin kropp som ett hinder för konception, vilket gav dem känslan av att vara ofullständiga i jämförelse med andra kvinnor. Sorgen över barnlösheten var ständigt närvarande hos kvinnorna. När kvinnorna accepterar sin situation som barnlös sker en personlig mognad och de börjar lägga fokus på sig själva och mål som är uppnåbara. Slutsatsen som framkommer i det här arbetet är att kvinnorna i den här litteraturöversikten oavsett kulturell bakgrund har liknande upplevelser i sin längtan efter ett barn. Att vara ofrivillig barnlös är en stor påfrestning för den som är drabbad. Det är av stor vikt att ofrivilligt barnlösa kvinnor erbjuds emotionellt stöd under sorgeprocessen som följer barnlösheten samt att stötta dem att identifiera strategier för att hantera sorgen.

Reproductive Medicine currently represents one of the fastest growing medical fields. The birth of Louise Joy Brown as the first baby conceived in vitro fertilization or in vitro, is considered being the historic milestone in development of the treatement methods in assised reproducion. Ethical discussions regarding the admissibility of these methods have developed as a result of technological advance of the infertility treatment. Adequate response of applicable law was required on the currant state of medicine which resulted in the need to regulate legally this field whether it's about establishing of assumption of fatherhood with regard to determination of fatherhood of a child conceived through artificial insemination, as well as determination of the basic conditions for the access to assisted reproductive techniques. Originally, the legislation of artificial insemination was defined only in the implementing regulation of the Ministry of Health, as the reproductive technologies were entirely new area. Today, the area of assisted reproduction is regulated in Act no. 373/2011. This thesis deals with the regulation of the assisted reproduction and it focuses on the controversial methods and procedures which are based on child sex selection, gamete donation and donor anonymity and also it deals with...

Objetivo: Avaliar as evidências disponíveis comparando a eficácia da estimulação ovariana (EO) com uso de citrato de clomifeno (CC) e/ ou letrozol (LTZ) para reduzir o consumo de FSH, em relação à estimulação ovariana padrão (EOP). Métodos: Realizamos uma revisão sistematizada e meta-análise de ensaios clínicos randomizados (ECRs) que compararam os desfechos reprodutivos na fertilização in vitro. As buscas foram realizadas em onze bancos de dados eletrônicos e avaliamos manualmente a lista de referência dos estudos incluídos e revisões similares. Nós estratificamos os resultados separando os estudos baseados no agente oral utilizado (CC ou LTZ) e nas características da mulher incluída (em que se espera e em que não se espera má resposta ovariana). Os desfechos avaliados foram risco relativo (RR) para nascimento vivo, gravidez clínica, aborto, e taxa de cancelamento de ciclo, Peto Odds Ratio (OR) para síndrome de hiperestímulo ovariano (SHO), e diferença média (MD) para número de óocitos captados e consumo de FSH (ampolas). Resultados: Foram incluídos 22 estudos nesta revisão. Considerando o grupo de mulheres em que se espera má resposta, a evidência sugere que o uso de CC durante a estimulação ovariana resulta em similares taxas de nascidos vivos (RR= 0,9, IC95% = 0,6 a 1,2, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,8 a 1,4, evidência de moderada qualidade); o uso de LTZ não causa alteração significativa no número de oócitos captados (MD= -0,4, IC95% = -0,9 a +0,1, evidência de alta qualidade). Considerando os estudos que avaliaram mulheres em que não se esperava má resposta, a evidência sugere que o uso de CC reduz o número de oócitos captados (MD= -4,6, IC95%= -6,1 a -3,0, evidência de alta qualidade) e o risco de SHO (Peto OR= 0,2, IC95%= 0,1 a 0,3, evidência de moderada qualidade), enquanto os resultados são semelhantes para taxas de nascidos vivos (RR= 0,9, IC 95% = 0,7 a 1,1, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,9 a 1,2, evidência de alta qualidade). Para os demais desfechos a qualidade das evidências foi baixa ou muito baixa. Conclusões: A utilização de CC em mulheres em que se espera má resposta tem a vantagem de alcançar resultados reprodutivos semelhantes com redução dos custos. Para as demais mulheres, o uso do CC tem a vantagem adicional de reduzir o risco de SHO, mas também reduz o número de oócitos captados. Mais estudos seriam necessários para avaliar o efeito do LTZ com o mesmo propósito. Estudos futuros devem ter como objetivo estudar a taxa de gravidez cumulativa por oócito captado, insatisfação da paciente e aceitação para repetir o ciclo se não engravidar, que são dados importantes para a tomada de decisões clínicas. / Objective: To assess the available evidence comparing effectiveness of ovarian stimulation (OS) using clomiphene citrate (CC) and/or letrozole (LTZ) for reducing FSH consumption compared with standard OS. Methods: We performed a systematic review and meta-analysis of randomized controlled trials (RCTs) that compared the reproductive outcomes following in vitro fertilization. We searched eleven electronic databases and hand-searched the reference list of included studies and related reviews. We stratified the results separating the studies depending on the oral agent (CC or LTZ) and on the characteristics of the included women (expected poor ovarian response or other women). When combining the results of included studies, we assessed the relative risk (RR) for live birth, clinical pregnancy, miscarriage, and cycle cancelation, Peto Odds Ratio (OR) for OHSS, and mean difference (MD) for the number of oocytes retrieved and FSH consumption. Results: A total of 22 studies were included in this review. Considering women with expected poor ovarian response, the available evidence suggests that using CC for reducing FSH consumption during OS provide similar live birth (RR=0.9, 95%CI=0.6-1.2, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.8-1.4, moderate quality evidence); the use of LTZ doesn\'t cause a relevant change on the number of oocytes retrieved (MD=-0.4, 95%CI= -0.9 to +0.1, high quality evidence). Considering the studies evaluating other women, the available evidence suggests that using CC for reducing FSH consumption during OS reduces the number of oocytes retrieved (MD=-4.6, 95%CI=-6.1 to -3.0, high quality evidence) and the risk of OHSS (Peto OR=0.2, 95%CI=0.1-0.3, moderate quality evidence), while results in similar live birth (RR=0.9, 95%CI=0.7-1.1, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.9-1.2, high quality evidence). The quality of the evidence was low or very low for the other outcomes. Conclusion: The use of CC for reducing FSH consumption in women with expected poor ovarian response has the advantage of providing similar reproductive outcomes with reduced costs. For the other women, the use of CC for reducing FSH consumption has the additional advantage of reducing OHSS, but also reduces the total number of oocytes retrieved. More studies are necessary to evaluate the effect of LTZ for the same purpose. Future studies should aim on cumulative pregnancy per oocyte retrieval, patient dissatisfaction and agreement to repeat the cycle if not pregnant; which are important outcomes for clinical decisions.

Orientadora: Marion Burkhardt de Koivisto / Banca: Gisele Zoccal Mingoti / Banca: Sony Dimas Bicudo / Resumo: O objetivo deste trabalho foi avaliar os efeitos da gota citoplasmática proximal (GCP) no sêmen de bovinos quanto à integridade do DNA, das membranas citoplasmática, acrossomal, no potencial mitocondrial e verificar a taxa de produção de embriões in vitro. Três amostras descongeladas de cinco (Controle: espermiograma normal), e oito touros Bos indicus (Gota: GCP ≥15%) foram avaliadas. Foram realizados os seguintes testes: motilidade e vigor pós-descongelação, concentração, morfologia espermática, teste de termo-resistência lento (TTL), integridade da membrana acrossomal, plasmática e potencial mitocondrial utilizando sondas fluorescentes (PI, FITC-PSA e JC-1) e integridade da cromatina pelo método de coloração com laranja de acridina. Dois touros com índices elevados de GCP e três animais controle foram selecionados para fertilização in vitro (FIV). As análises estatísticas foram efetuadas empregando-se o programa Statistical Analysis System. O nível de significância foi de 5%. Os resultados obtidos demonstraram que altos índices de GCP não afetaram a motilidade e o vigor, antes e após o TTL, assim como não interferiram na porcentagem de acrossomas intactos. Os resultados destas avaliações mostraram que a alta incidência de GCP afetou a integridade da membrana acrossomal e plasmática bem como a presença de potencial mitocondrial. No entanto, a alta incidência de GCP não promoveu aumento na porcentagem de injúrias à cromatina após descongelação, mas os resultados sugerem que podem ser mais sensíveis à desnaturação quando incubados por três horas. No experimento II, os índices de produção de embriões in vitro podem ter sido afetados pela interação da alteração morfológica e o efeito individual do touro. / Abstract: The objective of this study was to evaluate the effects of the proximal cytoplasmic droplets (PCD) in bovine semen, on the integrity of DNA, cytoplasmic membrane, acrossome, mitochondrial function and the rate of in vitro embryo production. Three batches of five (control group G1: normal sperm parameters) and eight Bos indicus bulls (G2: PCD ≥15%) were analysed. The following tests were carried out: post thaw motility and, vigor, concentration, sperm morphology, slow thermo-resistance (TRT), membrane integrity, acrossome status, mitochondrial function through fluorescent probes (FITC-PSA, PI and JC-1) and integrity of chromatin was accessed by acridine orange stain. Two bulls with high rates of PCD and three animals (control group) were selected for in vitro fertilization (IVF). Statistical analyses were performed using the Statistical Analysis System. The significance level was 5%. The results showed that high rates of PCD did not affect motility and vigor, before and after the TRT, and did not affect the percentage of intact acrossome. The results showed that the high incidence of PCD affected membrane integrity, acrossome status and mitochondrial function when compared to the G1 group due. However, the high incidence of PCD did not affect the percentage of chromatin injury after thawing, but results suggest that spermatozoa may be more susceptible to damage when incubated for three hours. In experiment II the embryo production rate may have been affected by the interaction of the morphology traits and the bull effect. / Mestre

The number of women diagnosed as infertile continues to grow every year. The psychological impact of the infertility experience has been said to create distress equivalent to that associated with life-threatening illnesses and has been linked with posttraumatic stress disorder (PTSD). Using shattered assumption and stress-buffering theory, this quantitative causal comparative study explored the potential functional relationship between infertility and PTSD. The majority of the (all-female) participants were 24- to 34-year-old college graduates. A 2 X 3 factorial between-subjects ANOVA examined and compared the cause and effect of the independent variables, fertility treatment and psychological intervention, on the dependent variable, PTSD. A multiple linear regression was conducted to understand PTSD symptomology scores. The results revealed that the type of infertility treatment does not impact PTSD symptomatology in medically diagnosed women. However, the main effect of psychological treatment was significant, as was infertility treatment by psychological treatment interaction. Additionally, the impact of fertility problems on the participant's physical health was the highest ranking predictor, which suggests that stress levels in women receiving infertility treatment are equivalent to those in women with cancer, AIDS, and heart disease, as suggested by other researchers. This study has implications for positive social change, in that it may promote better understanding of the psychological impact of infertility and decreased PTSD symptomatology for medically diagnosed infertile women. It opens the door for future research about the effectiveness of psychological intervention, and provides awareness of possible PTSD susceptibility.

La capacitación espermática puede ser mimetizada in vitro eliminando el plasma seminal por distintos sistemas de lavado. Entre los tratamientos espermáticos empleados habitualmente en los laboratorios para capacitar a los espermatozoides se encuentran los lavados que se realizan con medios enriquecidos con albúmina o a través de gradientes de Percoll. El objetivo de este trabajo ha sido determinar los cambios que acontecen en los espermatozoides (procedentes de epidídimo y eyaculados) sometidos a tres sistemas de capacitación in vitro evaluados mediante una batería de técnicas que determinan distintos estadios de la capacitación espermática.De los resultados obtenidos se desprende que tanto la procedencia de los espermatozoides (epidídimo o eyaculado) como el tratamiento de capacitación al que se les somete afecta en gran medida a los resultados de la penetración in vitro y por lo tanto, la capacitación se produce de manera diferente entre estos grupos. / Sperm capacitation may be defined as a set of molecular modifications that occurs in the spermatozoa, after maturation in the epididymis, which enables them to fertilize the oocyte. In vitro, this process can be imitated in vitro by separation of the seminal plasma by different systems of washing. Between the sperm treatments routinely used in the laboratories, semen samples are washed of albumin or centrifugations through a Percoll. The aim of this work was examine and characterize the changes that happen in the sperms (from epididymis and ejaculated) submitted to three systems of in vitro capacitation, evaluated by means of a battery of tests to determine different levels of the sperm capacitation. Sperm capacitation was dependent on sperm treatment, whetherepididymal or ejaculated and to whichever parameter measured.Nevertheless, all these parameters, in spite of the fact that they have beendescribed as tools to evaluate the sperm capacitation, really are not capable of discriminating or indicating the level of capacitation.

El objetivo de este trabajo consistió en describir el papel del sistema plasminógeno/plasmina (PLG/PLA) en la fecundación bovina y porcina. Mediante fecundación in vitro, demostramos que la presencia de PLG ó PLA en el medio de coincubación de los gametos disminuía la penetración de los espermatozoides en los ovocitos y su unión a la zona pelúcida (ZP). Esta disminución no se debía a alteraciones de la funcionalidad espermática ni a cambios en la resistencia de la ZP a la proteolisis, sino a que la PLA provocaba la liberación de los espermatozoides adheridos a la ZP. Mediante inmunofluorescencia indirecta detectamos la presencia de PLG y sus activadores en la ZP y en el oolema de los ovocitos antes de la fecundación. Tras la fecundación, dicha presencia disminuyó o desapareció por completo, por lo que proponemos que el sistema PLG/PLA se activa durante la interacción espermatozoide-ovocito y contribuye a regular la polispermia. / The aim of this study was to describe the role of the plasminogen/plasmin system (PLG/PLA) in bovine and porcine fertilization. Through in vitro fertilization, we demonstrated that the presence of PLG or PLA in the incubation medium of gametes decreased penetration of oocytes and sperm binding to the zona pellucida (ZP). This decrease was not due to alterations in sperm function or changes in the ZP resistance to proteolysis, but the PLA caused the release of sperm previously bound to the ZP. By indirect immunofluorescence we detected the presence of PLG and its activators in the ZP and oolema of the oocytes before fertilization. After fertilization, this presence diminished or disappeared completely, so we propose that the PLG/PLA system is activated during sperm-oocyte interaction and contributes to the regulation of polyspermy.

No description available.

Introdução: Novos medicamentos trouxeram um considerável aumento na chance de gravidez no momento em que a estimulação ovariana controlada permitiu aumentar o número de oócitos a serem recuperados e, consequentemente, um maior número de embriões para serem transferidos. Os análogos de hormônio liberador de gonadotrofina, em associação com gonadotrofinas, trouxeram a solução para um dos problemas da estimulação ovariana controlada: o pico precoce de hormônio luteinizante, que prejudicava a coleta de oócitos em torno de 20% dos casos. Rotineiramente, usamos o protocolo com agonista, mas a possibilidade do uso de antagonistas resulta em maior facilidade de manuseio pelo usuário, envolve um único menstrual ciclo e dispensa orientação para uso de drogas, no final do ciclo anterior. Assim, buscamos avaliar o uso dos dois protocolos e analisar seus resultados. Objetivos: Análise e comparação de dados entre dois protocolos de indução (longo com agonista e flexível com antagonista) em pacientes submetidas a técnicas de reprodução assistida na SEGIR – Serviço de Ecografica, Genética e Reprodução Assistida – Porto Alegre. Métodos: Estudo transversal comparando os resultados intermediários com o uso de dois diferentes protocolos de estimulação ovariana com de agonista versus antagonista do hormônio liberador de gonadotrofina para técnicas de reprodução assistida. A análise estatística dos dados recuperados (idade, índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados, dose total de FSH utilizada e ocorrência de síndrome do hiperestímulo ovariano) foi realizada através de teste t de Student para dados paramétricos e análise de covariância para as variáveis dependentes, calculados com o programa SPSS 16.0. Resultados: Um total de 50 pacientes preencheram os critérios para inclusão no estudo entre janeiro e março no ano de 2010, sendo 25 em cada grupo. Houve diferença estatística apenas na idade média entre os grupos (p=0,031). Não houve diferença estatística para os demais dados analizados (índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados e dose de FSH utilizada) entre os grupos. Não houve casos de síndrome do hiperestímulo ovariano. Conclusão: Ambos protocolos os são iguais em termos de resultados. O agonista tem vantagens sobre o agendamento do procedimento, mas leva muito tempo para começar a estimulação e tem a possibilidade de iniciar a medicação em uma paciente grávida. Somado a isso, temos a possibilidade de ter síndrome do hiperestímulo ovariano como complicação. No grupo antagonista, está claro a maior facilidade de uso da medicação e o início mais rápido da estimulação ovariana. / Background: New medications have brought a considerable increase in the chance of pregnancy at the time that controlled ovarian stimulation allowed an increase in the number of oocytes to be recruited and, consequently, a greater number of embryos to be transferred. The gonadotropin-releasing hormone analogues, in association with gonadotropins, brought the solution to one of the controlled ovarian stimulation's problems: the early peak of luteinizing hormone, which harmed the oocytes collection around 20% of the cases. We, routinely, used the agonist protocol, but the possibility of antagonists usage results in greater ease of handling by the user, involves a single menstrual cycle and dispensation guidance for drug use, at the end of the previous cycle. Thus we seek to evaluate the use of the two protocols and analyze their results. Objective: Analysis and compare data between two induction protocols (long agonist and flexible antagonist) in patients submitted an assisted reproduction technique in SEGIR – Serviço de Ecografia Genética e Reprodução assistida – Porto Alegre. Methods: Cross-sectional study comparing the intermediate results with the use of two different ovarian stimulation protocols with gonadotropin-releasing hormone agonist versus antagonist to assisted reproductive techniques. The statistical analysis of the retrieved data (age, body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved, total dose of FSH used and occurrence of ovarian hyperstimulation syndrome) was performed by Student t test for parametric data and analysis of covariance for the dependent variables, calculated with the program SPSS 16.0. Results: A total of 50 patients, 25 in each group, met the criteria for inclusion in the study between January and March in the year 2010. There was statistically significant difference only in the middle ages between the groups (p = 0 .031). There was no statistical difference for the remaining data analyzed (body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved and dose of FSH utilized) between the groups. There were no cases of ovarian hyperstimulation syndrome. Conclusion: Both protocols are equal in terms of results. The agonist has advantages about scheduling of the procedure, but it takes too long to start the stimulation and have possibility to start medication in a pregnant patient. Added to this, we have the possibility of getting the ovarian hyperstimulation syndrome as complication. In the antagonist group, is clear the ease-of-use of the medication and the fastest start of the ovarian stimulation.