One in six couples wishing to start a family are infertile. The many causes of infertility include genetic defects that can be single gene, multifactorial or chromosomal (including Y deletions, karyotype abnormalities and gamete aneuploidy). This thesis is concerned with the association between infertility and increased sperm aneuploidy. Specific questions are: should males be screened for sperm aneuploidy before intracytoplasmic sperm injection (ICSI)? Is there a relationship between individual semen parameters and sperm aneuploidy for specific chromosome pairs? What is the role of genome organisation in male gametes and its association with infertility? Whether use of alternative therapy (in this case, traditional Chinese Medicine (TCM)) can be used to improve sperm disomy levels. Statistical analysis of questionnaire data revealed that infertility specialists believed there to be merit in screening sperm aneuploidy levels before ICSI. Evidence is presented for possible chromosome-specific and semen parameter specific mechanisms for sperm aneuploidy as is evidence of genome organisation that may be perturbed in infertile males. Finally, in six males studied, sperm aneuploidy levels improved significantly coincident with TCM. Closer investigation of the biological activity of individual therapeutic herbs and treatment cocktails revealed strong anti-oestrogenic and anti-oxidant properties. This suggests a possible mechanism of action of the herbs and provides the basis from which future placebo controlled clinical trials might continue. Possible criticisms of the work presented here include the unavailability of blood samples from many of the patients (thus preventing karyotype analysis) and the absence of a second control group in our studies on semen parameters. Nevertheless significant steps have been made towards establishing the need for, and the implementation of, a pre-ICSI screening test. Moreover progress has been made towards further understanding the aetiology of sperm aneuploidy and towards the implementation of a new treatment that may, ultimately, augment, or even replace ICSI.

Tesis por compendio / [EN] In vitro production (IVP) of embryos has multiple applications, including the development of transgenic / genetically modified animals that are of broad utility in the biomedical field and could reach to be so in agriculture. In this sense, the intracytoplasmic sperm injection (ICSI) with transfected sperm, such as somatic cell nuclear transfer (SCNT) with transfected cells, have proved to be valid strategies to generate animals with stably integrated exogenous DNA, capable of transmitting to their offspring. However, to make it feasible the availability of a large number of IVP embryos it's necessary. Despite the development of IVP systems of pig embryos for these strategies in the last decades, and a high variety of protocols, there are still some critical issues that limit embryonic development and therefore its application on a large scale. The main objective of this thesis has then pursued, optimizing the ICSI and SCNT protocols in order to improve the viability of IVP porcine embryos. The initial approach was to address the study both in vitro maturation (IVM) and oocyte activation within the SCNT frame. To this end, the most efficient electrical activation protocol under our working conditions was selected from among three proposals, and we then studied the effect of serum supplement on IVM medium, noticing that it doesn't increase the development of partenogenetic embryos produced by electrical activation nor of those produced by SCNT. Moreover, and in order to optimize the ICSI technique, different aspects such as Ca2+ concentration in the microinjection medium, sperm pretreatment with Triton X-100 (TX) and the addition of cysteine or caffeine supplements to the in vitro culture (IVC) medium post-ICSI, were studied in order to promote sperm nucleus decondensation and oocyte activation. Noticing that: i) the concentration of Ca2+ did not influence the cleavage rate, or the development of partenogenic embryos from microinjected oocytes without sperm (sham injection); ii) caffeine did not increase either the pronuclear formation neither the subsequent embryo development, in combination with TX sperm pre-treatment it reduced oocyte activation and embryo development rates; iii) the pronuclear formation was not increased by the sperm pre-treatment with TX nor by the cysteine treatment, regardless of the time of the study assessment. / [ES] La producción in vitro (PIV) de embriones goza de múltiples aplicaciones, entre las que cabe destacar la obtención de animales transgénicos/modificados genéticamente que resultan de amplia utilidad en el ámbito biomédico y podrían llegar a resultarlo en el agropecuario. En este sentido, tanto la inyección intracitoplasmática de espermatozoides (ICSI) con espermatozoides transfectados, como la transferencia nuclear de células somáticas (SCNT) con células transfectadas, han demostrado ser estrategias válidas para generar animales con ADN exógeno integrado de manera estable, capaces de transmitirlo a su descendencia. Sin embargo, para lograrlo es necesario disponer de un gran número de embriones PIV. A pesar del desarrollo durante las últimas décadas de sistemas de PIV de embriones de porcino para estas estrategias, y la disponibilidad de gran variedad de protocolos, persisten aún ciertos puntos críticos que limitan el desarrollo embrionario y por lo tanto su aplicación a gran escala. El objetivo principal de esta tesis ha perseguido por lo tanto, la optimización de los protocolos de ICSI y SCNT con el fin de mejorar la viabilidad de los embriones PIV de porcino. El planteamiento inicial consistió en abordar el estudio tanto de la maduración in vitro (MIV) como de la activación oocitaria en el marco de la SCNT. Para ello se seleccionó de entre tres propuestas de activación artificial, el protocolo de activación eléctrica (que resultó ser el más eficiente bajo nuestras condiciones de trabajo), y se estudió a continuación el efecto del suplemento de suero en el medio de MIV, constatando que no se incrementaba el desarrollo de embriones partenogenotas producidos mediante activación eléctrica ni de aquellos producidos mediante SCNT. Por otra parte, y con el fin de optimizar la técnica de ICSI se abordaron diferentes aspectos como la concentración de Ca2+ en el medio de microinyección, el pre-tratamiento espermático con Triton X-100 (TX), así como la adición de suplementos de cafeína o cisteína al medio de cultivo in vitro (CIV) pos-ICSI, con el fin de favorecer la descondensación del núcleo espermático y la activación oocitaria. Observando que: i) la concentración de Ca2+ no influyó en la tasa de división, ni en la de desarrollo de embriones partenogenotas procedentes de oocitos microinyectados sin espermatozoide (sham injection); ii) la cafeína no incrementó la formación pronuclear ni en el desarrollo embrionario posterior, y en combinación con el pre-tratamiento espermático con TX redujo la activación oocitaria y el desarrollo embrionario; iii) la formación pronuclear no se vio incrementada por el pre-tratamiento espermático con TX ni por el tratamiento con cisteína, independientemente del momento de la evaluación estudiado. / [CA] La producció in vitro (PIV) d'embrions gaudeix de múltiples aplicacions, entre les quals cap assenyalar l'obtenció d'animals transgènics/modificats genèticament que resulten d'àmplia utilitat en el àmbit biomèdic y podrien arribar a ser-ho en el àmbit agropecuari. En aquest sentit, tant la injecció intracitoplasmàtica d'espermatozoïds (ICSI) amb espermatozoïds transfectats, com la transferència nuclear de cèl.lules somàtiques (SCNT) amb cèl.lules transfectades, han demostrat ser estratègies vàlides per a l'obtenció d'animals amb ADN exogen integrat de manera estable, capaços de transmetrela a la seva descendència. No obstant això, per aconseguir-ho és necessari disposar d'un gran nombre d'embrions PIV. Malgrat el desenvolupament durant les últimes dècades de sistemes de PIV d'embrions de porcí per a aquestes estratègies, i la disponibilitat de gran varietat de protocols, persisteixen encara certs punts crítics que limiten el desenvolupament embrionari i per tant la seva aplicació a gran escala. L'objectiu principal d'aquesta tesi ha perseguit per tant, l'optimització dels protocols d' ICSI i SCNT per tal de millorar la viabilitat dels embrions PIV de porcí. El plantejament inicial va consistir en abordar l'estudi tant de la maduració in vitro (MIV) com de l'activació oocitària en el marc de la SCNT. Per a això es va seleccionar entre tres propostes d'activació artificial, el protocol d'activació elèctrica (que va resultar ser el més eficient sota les nostres condicions de treball), i es va estudiar a continuació l'efecte del suplement de sèrum en el medi de MIV, constatant que no va incrementar el desenvolupament d'embrions partenogenotes produïts mitjançant activació elèctrica ni d'aquells produïts mitjançant SCNT. D'altra banda, i per tal d'optimitzar la tècnica de l'ICSI es van abordar diferents aspectes com la concentració de Ca2 + en el medi de microinjecció, el pretractament espermàtic amb Triton X-100 (TX), així com l'addició de suplements de cafeïna o cisteïna al mitjà de cultiu in vitro (CIV) pos-ICSI, per tal d'afavorir la descondensació del nucli espermàtic i l'activació oocitària. Observant que: i) la concentració de Ca2+ no va influir en la taxa de divisió, ni en la de desenvolupament d'embrions partenogenotes procedents d'oòcits microinjectats sense espermatozoïd (sham injection); ii) la cafeïna no va incrementar la formació pronuclear ni en el desenvolupament embrionari posterior, i en combinació amb el pretractament espermàtic amb TX va reduir l'activació oocitària i el desenvolupament embrionari; iii) la formació pronuclear no es va veure incrementada pel pretractament espermàtic amb TX ni pel tractament amb cisteïna, independentment del moment de l'avaluació estudiat. / García Mengual, ME. (2016). OPTIMIZACIÓN DE LAS ESTRATEGIAS REPRODUCTIVAS DE MICROMANIPULACIÓN PARA LA PRODUCCIÓN IN VITRO DE EMBRIONES PORCINOS [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61448 / Compendio

Des facteurs masculins sont identifiés dans près de la moitié des cas d’infertilité. À ce jour, les tests évaluant la fertilité masculine demeurent peu prédictifs de la survenue d’une grossesse. Dans le but de pallier cette lacune, nous avons mis au point deux nouveaux tests mesurant l’intégrité de l’ADN et le temps de survie des spermatozoïdes. Nous avons effectué une étude prospective portant sur 42 couples infertiles suivis en fécondation in vitro (FIV). Le spermogramme a été effectué selon les critères de l’Organisation Mondiale de la Santé (OMS) et le temps de survie des spermatozoïdes exposés à un détergent cationique a été mesuré en observant la mobilité sous microscope. L’intégrité de l’ADN des spermatozoïdes a été vérifiée par la nouvelle méthode de marquage radioenzymatique et par analyse de la structure de la chromatine (SCSA). Tous les tests ont été réalisés sur la partie des échantillons de sperme non utilisée par la clinique de fertilité. Le projet a été approuvé par le comité d’éthique du Centre Hospitalier Universitaire de Montréal (CHUM) et les patients ont préalablement signé un formulaire de consentement éclairé. L’analyse des paramètres du spermogramme et de l’intégrité de l’ADN n’a montré aucune différence statistiquement significative entre les données chez les couples avec ou sans grossesse. Cependant, le taux de grossesse biochimique était statistiquement plus élevé chez les couples dont le temps de survie des spermatozoïdes était long (>250 s) comparativement à ceux dont ce temps était court (≤250 s): 66% vs 27% respectivement (p<0,05). Les taux de grossesse clinique et d’implantation étaient aussi plus élevés, mais les différences n’atteignaient pas le seuil de signification statistique. Nos résultats confirment que le spermogramme et la mesure de la fragmentation de l’ADN des spermatozoïdes ne sont pas de bons facteurs prédictifs des résultats de la FIV. Par contre, le test de survie des spermatozoïdes serait un meilleur indicateur de la possibilité d’une grossesse en FIV. L’amélioration de sa spécificité et un plus grand nombre de sujets sont nécessaires avant de proposer son application en clinique de fertilité. / Male factors are known to be involved in almost half of the couples consulting for infertility. To date, the tests for evaluating male fertility are poor predictors of pregnancy. We developed two new tests to evaluate sperm function: a sperm survival test and a new method to measure sperm DNA integrity. This prospective study was conducted on 42 infertile couples undergoing in vitro fertilization (IVF). Assessment of sperm parameters was done according to the World Health Organization (WHO) criteria, and sperm survival upon exposure to a cationic detergent was measured by observing motility under the microscope. Sperm DNA integrity was verified by our new radioenzymatic method as well as by the sperm chromatin structure analysis (SCSA) method. All testing was performed on a remainder aliquot of the semen samples. The study was approved by the ethics committee of the Centre Hospitalier Universitaire de Montréal (CHUM), and informed consent was obtained before inclusion. Neither conventional semen analysis, nor sperm DNA fragmentation showed statistically significant difference between conception and non-conception cycles. However, the biochemical pregnancy rate was statistically higher in couples where the sperm survival time was long (>250 s) compared to short (≤250 s): 66% vs. 27% respectively, (p < 0.05). The clinical pregnancy rate and implantation rate were also higher but the differences did not reach statistical significance. Our study confirms that conventional semen analysis and the assay for sperm DNA integrity are not reliable indicators of IVF outcome. In contrast, our new sperm survival test seems to be a better predictor of the pregnancy rate after IVF. Improvement of its specificity and a larger cohort of patients are necessary before proposing its regular application in IVF clinics.

Justificativa: Falhas recorrentes de implantação (FRI) são uma fonte de grande frustração para pacientes e especialistas que, frequentemente buscam intervenções com o objetivo de atingir resultados favoráveis. A prevalência exata dessa condição é de difícil estimativa, uma vez que existem diversas definições para caracterizá-la. Diversas intervenções que visam a melhora dos resultados reprodutivos dessas pacientes já foram propostas. Entretanto, nenhuma revisão sistematizada abordou, simultaneamente, todas as potenciais estratégias para esse grupo de mulheres. Dessa forma, a eficácia e a segurança dessas intervenções não são bem definidas. Devido ao alto número de intervenções descritas para esse fim, julgamos que uma metanálise que as contemple de forma abrangente é importante para casais, especialistas e pesquisadores do assunto. Objetivos: Avaliar a eficácia e segurança das intervenções que visam a melhora do resultado reprodutivo das pacientes com FRI submetidas a um novo tratamento de reprodução assistida. Métodos de busca: As buscas por estudos randomizados e controlados, publicados e em andamento, foram realizadas nas principais bases de dados eletrônicas. Adicionalmente, as listas de referências de estudos incluídos e revisões semelhantes foram avaliadas pelos autores. A última busca eletrônica foi realizada em fevereiro de 2018. Critérios de Elegibilidade: Foram considerados elegíveis apenas os estudos verdadeiramente randomizados que comparassem quaisquer intervenções destinadas a esse grupo depacientes. No presente estudo, consideramos como FRI a história de duas ou mais falhas prévias. Extração e análise de dados: Dois autores realizaram, individualmente, a seleção de estudos, extração de dados e análise do risco de viés. Discordâncias foram resolvidas em consulta a um terceiro autor. Os pesquisadores de estudos potencialmente elegíveis foram contatados sempre que necessário para obtenção de informações adicionais. Resultados: Foram identificados 2794 registros; desses, 62 estudos foram incluídos, representando uma população de 9308 pacientes, submetidas a 26 intervenções diferentes. Informações sobre 24 intervenções e 8461 pacientes foram submetidas à análise quantitativa. Não há evidências de alta ou moderada qualidade de que alguma dessas intervenções seja realmente eficaz para a melhora dos resultados reprodutivos de pacientes com FRI. As seguintes intervenções se mostraram benéficas: assisted hatching, injúria endometrial, histeroscopia, uso de FSH urinário + recombinante para estimulação endometrial e administração intrauterina de hCG. Porém as evidências são de baixa qualidade, o que nos traz incerteza em relação aos seus reais efeitos. Todas as outras intervenções identificadas não permitiram nenhuma outra conclusão adicional, uma vez que as evidências foram avaliadas como de muito baixa qualidade ou não foram encontrados estudos randomizados que as tivessem avaliado. Conclusões: Evidências de ensaios clínicos randomizados não sustentam o uso de nenhuma intervenção para a melhora dos resultados reprodutivos de pacientes com FRI. Todos os achados foram julgados como de baixa ou muito baixa qualidade, o que nos traz incerteza quanto aos seus reais efeitos na prática clínica. Deve-se estar ciente de que o emprego de tais intervenções impõe despesas e riscos adicionais para as pacientes. Além disso, a falta de critérios universalmente aceitos para odiagnóstico de falha recorrente de implantação é uma importante limitação para o avanço do conhecimento sobre essa condição / Background: Recurrent implantation failure (RIF) is a source of deep frustration to couples and clinicians, who often look for interventions to improve the reproductive outcomes. Its exact prevalence is difficult to determine because there are several definitions used to describe the condition. Several interventions aiming to improve reproductive outcomes for such patients have been studied. However, there are no systematic reviews that focus on all potential interventions for improving reproductive outcomes in women with RIF undergoing assisted reproduction techniques. The efficacy and safety of these interventions are not clear. Because of the large number of potential interventions for this condition, it would be very difficult to be aware of the current evidence for all of them. We believe this systematic review is important for subfertile couples, clinicians and researchers. Objectives: To assess the efficacy and safety of interventions designed to improve reproductive outcomes in women with RIF undergoing ART. Search methods: We searched for randomised controlled trials (RCT) in electronic databases (Cochrane Gynaecology and Fertility Group (CGF), The Cochrane Central Register of Controlled Trials, MEDLINE Ovid, EMBASE Ovid, PsycINFO, PsycINFO Ovid, CINAHL, LILACS), trials registers (ClinicalTrials.gov, ISRCTN registry, The WHO International Clinical Trials Registry Platform, World Health Organization International Clinical Trials Registry Platform) and grey literature (OpenGrey); in addition, we handsearched the reference lists of included studies and similar reviews. We performed the last electronic search on 22 Feb 2018.Selection criteria: We considered eligible only truly randomised controlled trials comparing any intervention designed to improve outcomes in women with repeat implantation failure (RIF) compared to other intervention, placebo or no treatment. For study selection, we defined RIF as two or more previous failures Data collection and analysis: Two authors independently performed study selection, data extraction, and assessment of the risk of bias. Any disagreements were solved by consulting a third review author. Study\'s authors were contacted whenever needed to solve any queries. Results: the search retrieved 2794 records; from those, sixty-two studies were included, comprising 9308 participants, submitted to 26 different interventions. Data from 24 interventions and 8461 participants were pooled for quantitative analysis. We found no high or even moderate quality evidence that any of the tested interventions are really effective to improve reproductive outcomes of women with RIF undergoing a new IVF treatment. We observed low-quality evidence of benefit for women with RIF with the following interventions: assisted hatching, endometrial Injury, hysteroscopy, the use of human + recombinant FSH for ovarian stimulation and intrauterine hCG administration. All the other listed interventions did not allow any further conclusion: either very low-quality evidence or no evidence from RCTs. Conclusions: Evidence from RCTs does not support the use of any specific intervention for improving reproductive outcomes in women with RIF. All evidences were deemed of low to very low quality, which makes us uncertain of their real effectiveness on clinical practice. One should be aware that the employment of such interventions imposes additional expenses and risks. Additionally, the lack of universally accepted criteria for recurrent implantation failure is an important limitation for the advance of knowledge regarding this condition.More studies are needed to evaluate their real effect. Maybe even more importantly is to create universally accepted criteria for defining implantation failure; only them one will be able to test interventions for this specific group

Des facteurs masculins sont identifiés dans près de la moitié des cas d’infertilité. À ce jour, les tests évaluant la fertilité masculine demeurent peu prédictifs de la survenue d’une grossesse. Dans le but de pallier cette lacune, nous avons mis au point deux nouveaux tests mesurant l’intégrité de l’ADN et le temps de survie des spermatozoïdes. Nous avons effectué une étude prospective portant sur 42 couples infertiles suivis en fécondation in vitro (FIV). Le spermogramme a été effectué selon les critères de l’Organisation Mondiale de la Santé (OMS) et le temps de survie des spermatozoïdes exposés à un détergent cationique a été mesuré en observant la mobilité sous microscope. L’intégrité de l’ADN des spermatozoïdes a été vérifiée par la nouvelle méthode de marquage radioenzymatique et par analyse de la structure de la chromatine (SCSA). Tous les tests ont été réalisés sur la partie des échantillons de sperme non utilisée par la clinique de fertilité. Le projet a été approuvé par le comité d’éthique du Centre Hospitalier Universitaire de Montréal (CHUM) et les patients ont préalablement signé un formulaire de consentement éclairé. L’analyse des paramètres du spermogramme et de l’intégrité de l’ADN n’a montré aucune différence statistiquement significative entre les données chez les couples avec ou sans grossesse. Cependant, le taux de grossesse biochimique était statistiquement plus élevé chez les couples dont le temps de survie des spermatozoïdes était long (>250 s) comparativement à ceux dont ce temps était court (≤250 s): 66% vs 27% respectivement (p<0,05). Les taux de grossesse clinique et d’implantation étaient aussi plus élevés, mais les différences n’atteignaient pas le seuil de signification statistique. Nos résultats confirment que le spermogramme et la mesure de la fragmentation de l’ADN des spermatozoïdes ne sont pas de bons facteurs prédictifs des résultats de la FIV. Par contre, le test de survie des spermatozoïdes serait un meilleur indicateur de la possibilité d’une grossesse en FIV. L’amélioration de sa spécificité et un plus grand nombre de sujets sont nécessaires avant de proposer son application en clinique de fertilité. / Male factors are known to be involved in almost half of the couples consulting for infertility. To date, the tests for evaluating male fertility are poor predictors of pregnancy. We developed two new tests to evaluate sperm function: a sperm survival test and a new method to measure sperm DNA integrity. This prospective study was conducted on 42 infertile couples undergoing in vitro fertilization (IVF). Assessment of sperm parameters was done according to the World Health Organization (WHO) criteria, and sperm survival upon exposure to a cationic detergent was measured by observing motility under the microscope. Sperm DNA integrity was verified by our new radioenzymatic method as well as by the sperm chromatin structure analysis (SCSA) method. All testing was performed on a remainder aliquot of the semen samples. The study was approved by the ethics committee of the Centre Hospitalier Universitaire de Montréal (CHUM), and informed consent was obtained before inclusion. Neither conventional semen analysis, nor sperm DNA fragmentation showed statistically significant difference between conception and non-conception cycles. However, the biochemical pregnancy rate was statistically higher in couples where the sperm survival time was long (>250 s) compared to short (≤250 s): 66% vs. 27% respectively, (p < 0.05). The clinical pregnancy rate and implantation rate were also higher but the differences did not reach statistical significance. Our study confirms that conventional semen analysis and the assay for sperm DNA integrity are not reliable indicators of IVF outcome. In contrast, our new sperm survival test seems to be a better predictor of the pregnancy rate after IVF. Improvement of its specificity and a larger cohort of patients are necessary before proposing its regular application in IVF clinics.

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.

The follicle and its enclosed oocyte share intimate and critical communication that regulates folliculogenesis and produces a mature oocyte. Protein and RNA accumulated in the oocyte during oogenesis control fertilization and direct embryonic development until the embryonic genome activates. Most knowledge of mammalian oocyte biology has been derived from eutherian species. Marsupials deserve more detailed studies because they have a distinct reproductive biology that offers a unique perspective from which to consider mammalian reproduction. The oocyte biology of the tammar wallaby, Macropus eugenii, is the focus of research in this thesis. Cold storage, a simple method for transporting ovarian tissue, was evaluated using histological techniques and follicle culture to assess the structure and function of tammar ovarian tissue. In vitro techniques were used to examine and compare: -folliculogenesis in prepubertal and adult animals, - fertilization of in vivo and in vitro matured oocytes, - and embryo development in follicular and tubal oocytes. / Tammar ovaries were placed in cold storage (PBS at 4?C) for 24 or 48 hours. Necrotic changes were minimal in ovarian follicles after cold storage and preantral follicles isolated from ovarian tissue after cold storage grew by similar amounts as non-stored follicles when cultured for 4 days in vitro. Although the general morphology and growth of follicles are unaffected after cold storage for up to 48 hours, the viability of the oocyte is of prime importance. The next important stages of this study were to develop in vitro techniques for follicle culture and for oocyte maturation and fertilization for future assessment of oocytes after cold storage. / A defined (serum-free) culture system was developed to grow isolated preantral follicles from prepubertal and adult tammars. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. Although FSH promoted growth in follicles from adult tammars, other factors present in serum were required for antrum formation. Therefore, once the hypothalmo-pituitary-gonadal axis is mature, hormones and growth factors modify the mechanism of antrum formation. Only follicles that developed an antrum in the presence of serum had granulosa and theca layers that had appropriately differentiated. While FSH stimulates follicle growth in vitro, more complex conditions are required to promote granulosa and theca differentiation. / Intra-cytoplasmic sperm injection (ICSI) was successfully used to compare fertilization of in vivo and in vitro matured oocytes as well as the development of mature oocytes collected from the ovary (surrounded by zona pellucida) or from the oviduct (surrounded by zona pellucida and mucoid coat). In vitro matured oocytes proceeded though the early stages of fertilization (e.g. sperm nuclear decondensation, pronuclear formation), but not syngamy. After sperm injection, in vivo matured oocytes cleaved as far as the 8-cell stage. Oocytes do not lose their ability to fertilize after acquisition of the mucoid coat, since tubal oocytes cleaved as far as the 8-cell stage after sperm injection. Follicular oocytes develop as far as the 5-cell stage after sperm injection, but embryos had a large cleavage cavity that hindered cell-cell contact. While the mucoid coat is not required for cleavage, it is important for appropriate cell-cell interaction and normal early development of the embryo. / This, the most detailed in vitro study of marsupial oocyte biology, has shown that there are many similarities in the biology of marsupial and eutherian oocytes but that the unique biology of marsupials offers a significant perspective on mammalian reproduction. This work also lays the foundation for the effective use of assisted reproductive techniques for conservation of Australia’s unique mammalian fauna.

L'objectif de ce travail est d'approfondir l'exploration du génome et de l'épigénome somatique et germinal chez des hommes présentant une atteinte sévère de la spermatogenèse. <br />Concernant le génome somatique, nous avons recherché la présence de mosaïques somatiques pour le chromosome Y microdélété chez 44 hommes infertiles, et aucune mosaïque n'a été détectée. Nous avons également analysé des réarrangements complexes du chromosome Y chez 3 patients infertiles. Cette étude nous a permis d'affiner leur caryotype et de mieux définir l'implication de l'anomalie du Y dans le phénotype d'infertilité. La ségrégation méiotique des chromosomes X, Y, 18, 13 et 21 a été étudiée par FISH multi-couleurs sur les spermatozoïdes de 31 patients infertiles. Nous avons montré des taux de disomies spermatiques augmentés chez la moitié de ces patients et identifié 4 facteurs de risques cliniques ou biologiques associés à l'augmentation des anomalies chromosomiques spermatiques. Enfin, concernant l'exploration de l'épigénome germinal, nous avons caractérisé le profil d'acétylation des histones par immunohistochimie sur les biopsies testiculaires de 33 patients atteints d'un syndrome des cellules de Sertoli isolées et/ou d'une tumeur testiculaire. Nous avons mis en évidence une hyperacétylation globale massive du noyau des cellules de Sertoli lorsque les tubes séminifères sont dépourvus de cellules méiotiques et post-méiotiques. Cette étude a révélé que l'acétylation des histones pourrait être impliquée dans le dialogue entre cellules germinales et cellules de Sertoli, et que sa dérégulation pourrait être associée à la genèse des cancers testiculaires. Ce travail ouvre des perspectives intéressantes pour la prise en charge des infertilités masculines sévères.

The follicle and its enclosed oocyte share intimate and critical communication that regulates folliculogenesis and produces a mature oocyte. Protein and RNA accumulated in the oocyte during oogenesis control fertilization and direct embryonic development until the embryonic genome activates. Most knowledge of mammalian oocyte biology has been derived from eutherian species. Marsupials deserve more detailed studies because they have a distinct reproductive biology that offers a unique perspective from which to consider mammalian reproduction. The oocyte biology of the tammar wallaby, Macropus eugenii, is the focus of research in this thesis. Cold storage, a simple method for transporting ovarian tissue, was evaluated using histological techniques and follicle culture to assess the structure and function of tammar ovarian tissue. In vitro techniques were used to examine and compare: -folliculogenesis in prepubertal and adult animals, - fertilization of in vivo and in vitro matured oocytes, - and embryo development in follicular and tubal oocytes. / Tammar ovaries were placed in cold storage (PBS at 4?C) for 24 or 48 hours. Necrotic changes were minimal in ovarian follicles after cold storage and preantral follicles isolated from ovarian tissue after cold storage grew by similar amounts as non-stored follicles when cultured for 4 days in vitro. Although the general morphology and growth of follicles are unaffected after cold storage for up to 48 hours, the viability of the oocyte is of prime importance. The next important stages of this study were to develop in vitro techniques for follicle culture and for oocyte maturation and fertilization for future assessment of oocytes after cold storage. / A defined (serum-free) culture system was developed to grow isolated preantral follicles from prepubertal and adult tammars. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. Although FSH promoted growth in follicles from adult tammars, other factors present in serum were required for antrum formation. Therefore, once the hypothalmo-pituitary-gonadal axis is mature, hormones and growth factors modify the mechanism of antrum formation. Only follicles that developed an antrum in the presence of serum had granulosa and theca layers that had appropriately differentiated. While FSH stimulates follicle growth in vitro, more complex conditions are required to promote granulosa and theca differentiation. / Intra-cytoplasmic sperm injection (ICSI) was successfully used to compare fertilization of in vivo and in vitro matured oocytes as well as the development of mature oocytes collected from the ovary (surrounded by zona pellucida) or from the oviduct (surrounded by zona pellucida and mucoid coat). In vitro matured oocytes proceeded though the early stages of fertilization (e.g. sperm nuclear decondensation, pronuclear formation), but not syngamy. After sperm injection, in vivo matured oocytes cleaved as far as the 8-cell stage. Oocytes do not lose their ability to fertilize after acquisition of the mucoid coat, since tubal oocytes cleaved as far as the 8-cell stage after sperm injection. Follicular oocytes develop as far as the 5-cell stage after sperm injection, but embryos had a large cleavage cavity that hindered cell-cell contact. While the mucoid coat is not required for cleavage, it is important for appropriate cell-cell interaction and normal early development of the embryo. / This, the most detailed in vitro study of marsupial oocyte biology, has shown that there are many similarities in the biology of marsupial and eutherian oocytes but that the unique biology of marsupials offers a significant perspective on mammalian reproduction. This work also lays the foundation for the effective use of assisted reproductive techniques for conservation of Australia’s unique mammalian fauna.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-23Bitstream added on 2014-06-13T20:24:31Z : No. of bitstreams: 1 borgesjunior_e_dr_botfm.pdf: 275485 bytes, checksum: 68fdbf94b0908ce788462bae7d5d1161 (MD5) / Cpdp - Centro Paulista de Pesquisa e Diagnostico / Desde o primeiro relato de sucesso em humanos, a Injeção Intracitoplasmática de Espermatozóide (ICSI) tem sido particularmente indicada para casos de alterações seminais graves, sendo já demonstrada uma relação diretamente proporcional entre os resultados deste procedimento e a qualidade seminal. Tem sido sugerido que a incapacidade de o espermatozóide iniciar a ativação oocitária seja uma das principais causas de falha de fertilização após a ICSI. Estudos prévios identificaram o cálcio como um importante segundo mensageiro durante a ativação oocitária e que o tratamento com ionoforo do calcio e capaz de favorecer a ativação oocitária, resultando em fertilização, desenvolvimento embrionário e gestações normais. O objetivo deste estudo foi avaliar o efeito da Ativação Oocitária Artificial (AOA) com ionoforo do calcio A23187 em ciclos de ICSI quando utilizados espermatozóides de diferentes origens. Foram avaliados 314 ciclos de ICSI divididos em tres Grupos: EJACULADO (n = 92), EPIDIDIMARIO (n = 82) e TESTICULAR (n = 140), sendo cada um deles dividido em subgrupos, dependendo da realizacao (Subgrupo AOA) ou nao de AOA (Subgrupo Controle . CT). Foram avaliados tambem separadamente os ciclos de mulheres com idade inferior a 36 anos, objetivando minimizar o efeito da idade sobre os resultados dos tratamentos. Para a ativação oocitária, os oócitos foram mantidos por 30 minutos apos a ICSI em meio de cultivo para AOA, contendo 5ÊM de ionoforo de calcio A23187 e em aproximadamente 20 horas a fertilizacao foi confirmada pela presenca de 2 Pro-Nucleos. Para cada grupo experimental foram comparados, entre os Subgrupos AOA e CT, os seguintes parametros: taxa de fertilizacao normal, taxa de falha parcial de fertilizacao, porcentagem de bons embrioes no dia da transferencia, taxa de gestacao clinica, taxa de implantacao e taxa de abortamento... / Since the first report of a birth after ICSI, it has been specially used for severe male factor of infertility and it was demonstrated that the semen quality influences the ICSI outcomes. Previous studies suggested that failure of fertilization after ICSI may be due to a spermatozoa inability in trigger the oocyte activation. Calcium has long been identified as a universally important second messenger during oocyte activation and many studies demonstrated that treatment with calcium ionophore may increase the free intracellular calcium, promoting oocyte activation, resulting in fertilization, embryo development and pregnancies. The aim of this study was to evaluate the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 in ICSI cycles using sperm from different origins. It was evaluated 314 ICSI cycles divided in groups according to the origin of spermatozoa: EJACULATED group (n = 92), EPIDIDYMAL group (n = 82) and TESTICULAR group (n = 140). Each group was split into sub-groups depending on the AOA treatment: sub-group AOA, when it was performed AOA and sub-group Control-CT, when it was not performed AOA. Furthermore, it was evaluated separately only cycles in each woman were younger then 36 years old. After ICSI, oocytes were incubated in culture medium containing calcium ionophore A23187 (5ìM concentration) for 30 minutes and in approximately 20 hours oocytes were checked for normal fertilization, defined as observation of two distinct pronucleous. For each experimental group the following parameters were compared between the sub-groups AOA and CT: normal fertilization rate; partial fertilization fail rate; percentage of high quality embryos on the transfer day; implantation rate; pregnancy rate and miscarriage rate. For all the evaluated parameters, it was not observed any significant difference between the subgroups for the three spermatozoa origin groups.... (Complete abstract click electronic access below)