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Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-17T11:52:18Z No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-17T11:54:08Z (GMT) No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-17T11:54:08Z (GMT). No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-09-20 / Aims: To make a review article on ectopic pregnancy (EP) in high complexity fertilization, to define the epidemiological and reproductive profile of women carriers of EP, to evaluate the prevalence of EP after in vitro fertilization (IVF) and to verify if the transfer of frozen embryos reduces the rates of EP. METHODS: All fresh and frozen embryos transfers obtained through IVF via ICSI conducted between January 2007 and December 2014 were reviewed. Clinical pregnancies characterized by the visualization of an intrauterine gestational sac or ectopic pregnancy were included in the evaluation. Ectopic pregnancies were diagnosed by ultrasonography and / or laparoscopy. The prevalence of EP and the comparison of the rates between fresh and frozen embryos transfers were analyzed by logistic regression using the SPPS software version 15.0. Results: Of the 933 embryos obtained through IVF that resulted in clinical pregnancies, 19 cases of EP were found. The fertilization with fresh embryos resulted in 772 clinical pregnancies (group I), and 161 with frozen embryos (group II). The global prevalence of EP was 2.02%. When considered by group, group one had 16 EP that represented a rate of 2.1% and group two had a rate of 1.9%. There was a lower rate of ectopic pregnancy with frozen embryos in comparison to fresh embryos, although this did not demonstrate statistical significance (p=0.86)(OR=0.89, IC=0.258-3.11). CONCLUSIONS: The reproductive profile of patients that exhibit EP was of young female with young partner, good ovarian response and that had more than 3 embryos transferred. The prevalence of EP in this study was 2.02. It was observed that there was no significant difference in the occurrence of EP when both groups, frozen and fresh embryos, were compared. / Objetivos: fazer um artigo de revisão sobre gestação ectópica (GE) em fertilização de alta complexidade, definir o perfil epidemiológico e reprodutivo das mulheres portadores de GE, avaliar a prevalência de GE pós-fertilização de alta complexidade e verificar se a transferência de embriões congelados reduz as taxas de GE. Métodos: foram revisadas todas as transferências de embriões frescos e congelados realizadas através de fertilização in vitro pela técnica de injeção intracitoplasmática de espermatozoides no período compreendido entre janeiro de 2007 a dezembro de 2014. Foram incluídas as gestações clínicas, sendo consideradas as gestações com visualização de saco gestacional intraútero e gestações ectópicas. As gestações ectópicas foram diagnosticadas por ultrassonografia e/ou laparoscopia. A prevalência de ectópica e a comparação das taxas entre os grupos de embriões frescos e congelados foram verificadas por meio da análise de regressão logística utilizando o programa Software SPSS versão 15.0. Resultados: foram analisadas 933 fertilizações in vitro que resultaram em gestações clínicas e verificou-se 19 casos de gestações ectópicas. As fertilizações com embriões frescos totalizaram 772 (grupo I) e com embriões congelados 161 (grupo 2). A prevalência de gestação ectópica foi de 2,02 %. No grupo I ocorreram 16 gestações ectópicas representando uma taxa de 2,1 % das fertilizações, no grupo 2 a taxa foi de 1,9 %. Uma menor taxa de gestação ectópica com embriões congelados foi verificada em comparação com embriões frescos, porém, não apresentou significância estatística (p=0,86) (OR= 0,89, IC 0,258 – 3,11). Conclusões: o perfil reprodutivo das pacientes que apresentaram GE foi de mulheres jovens com parceiros jovens, com boa resposta ovariana e que tiveram mais de três embriões transferidos. A prevalência de GE verificada no presente estudo foi de 2,02 %. Observou-se que não houve diferença significativa quanto à GE quando foram comparadas as transferências de embriões frescos com os congelados.

In Deutschland unterliegt die Reproduktionsmedizin umfassenden gesetzlichen Regelungen. Fertilisierte Oozyten müssen im Pronukleus-Stadium selektiert werden, hierbei darf maximal eine Anzahl von drei Embryonen kultiviert werden. Studien der vergangenen Jahre zielten vornehmlich auf die Entwicklung eines detaillierten Scoring-Systemes (Zygoten Screening im Pronukleus Stadium), um jeweils die Embryonen mit dem größten Entwicklungspotenzial zu selektieren. 99 Patientinnen wurden inkludiert und durchliefen entweder eine IVF oder ICSI Prozedur. Die fertilisierten Oozyten wurden im Pronukleus-Stadium beurteilt. TNF alpha und LIF, beides in der Reproduktionsmedizin bekannte Zytokine, wurden in gepoolten Kulturmedien an den Tagen 3 und 5 gemessen. 865 Oozyten wurden hierbei gewonnen, 438 zeigten positive Fertilisations-Zeichen, es fand sich eine Fertilisationsrate von 62,6%. Die Schwangerschaftsrate betrug 24,7%. Die mittlere PN Scores zeigten sich signifikant niedriger bei nicht konzipierenden Frauen (15.8 versus 17.2). Die mittlere TNF alpha Konzentration zeigte sich sowohl an Tag 3 als auch an Tag 5 signifikant erniedrigt in schwangeren Frauen gegenüber denen, welche nicht konzipierten (0.43pg/ml versus 0.59pg/ml). Die mittlere LIF Konzentration hingegen war signifikant erhöht bei schwangeren Frauen (56.2pg/ml versus 22.2pg/ml an Tag 3). Zusammenfassung: Das PN-Scoring bleibt eine gute Methode zur prognostischen Einschätzung des weiteren Entwicklungspotenziales von Präimplantationsembryonen. Höhere Konzentrationen von LIF und niedrigere Konzentrationen von TNF alpha in Kulturmedien scheinen eine favorable Rolle in der Embryogenese zu spielen. / German law comprehensively regulates IVF procedures and the selection of embryos for further cultivation. By law, fertilized oocytes have to be selected at the pronucleus-stadium, and a maximum of three embryos are allowed to be cultivated. Studies of the past years therefore aimed to elaborate a detailed scoring system (zygote scoring system at the pronucleus stage) in order to select those embryos with the highest potential for further favorable development. METHODS: 99 patients were included in our prospective trial and underwent either IVF or ICSI procedure. Fertilized oocytes were assessed at the pronucleus stage (day 1). TNF alpha and LIF, both cytokines known to be involved in embryonic development, were measured in pooled culture media on day 3 and 5. RESULTS: A total of 865 oocytes were collected, 438 showed positive signs of fertilisation, a fertilisation-rate of 62.6 % was achieved. Pregnancy rate per embryo transfer was 24.7 %. Mean PN-scores were significantly lower in women conceiving (15.8) than in those not conceiving (17.2). Mean TNF alpha concentration in culture media on day 3 was 0,54pg/ml and 0.37pg/ml on day 5 and was significantly lower in women conceiving (0.43pg/ml versus 0.59pg/ml on day 3). Mean LIF concentration on day 3 was 31.5pg/ml and 35.5pg/ml on day 5 and was significantly higher in women conceiving (56.2pg/ml versus 22.2pg/ml on day 3). CONCLUSION: PN-scoring remains a valuable tool for assessing fertilized oocytes for their further developmental potential at the pronucleus stage. Higher concentrations of LIF and lower concentrations of TNF alpha in culture media seem to play a putative favorable role in embryogenesis.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Etwa 15% aller Paare bleiben ungewollt kinderlos. Männliche Faktoren sind in circa einem Drittel der Fälle als ursächlich anzusehen. Jedoch sind die Erfolgsraten der Therapie männlicher Infertilität durch assistierte Reproduktion auch nach über 30 Jahren seit deren Einführung unbefriedigend. Bestehende Spermienaufbereitungsmethoden wie einfaches Waschen, swim up oder die Dichtegradientenzentrifugation basieren auf makroskopisch-funktionellen Parametern wie Motilität und Morphologie. Spezifische Eigenschaften wie etwa eine aktivierte Apoptosesignalkaskade der Spermien werden dabei nicht berücksichtigt. Die wesentlichen Elemente verschiedener Signalwege der aus somatischen Zellen bekannten Apoptose konnten auch am humanen ejakulierten Spermatozoon nachgewiesen werden. Über die (negativen) Auswirkungen der Apoptose auf die männliche Fruchtbarkeit gibt es einen Konsens. Ziel der vorliegenden Arbeit war es, Selektionsmethoden zu entwickeln, welche auf subzellulärer Ebene intakte Spermien mit dem größtmöglichen Fertilisationspotential aus dem Ejakulat extrahieren. In einem ersten Versuchskomplex konnte gezeigt werden, dass durch die Kombination von Dichtegradientenzentrifugation und swim-up (Standardmethoden in Reproduktionskliniken und andrologischen Laboren) zur Aufbereitung der Spermien von subfertilen Patienten eine akzeptable Reduktion der Spermien mit aktivierter Apoptosesignalkaskade erreicht werden kann. Jedoch gaben die großen interindividuellen Unterschiede im Separationseffekt Anlass zur Entwicklung innovativer Untersuchungs- und Separationsmethoden. So wurden unter anderem fluoreszenzbasierte Tests zur Evaluation von Spermiendefekten, wie beispielsweise einer gestörten Integrität des mitochondrialen Membranpotentials, eingeführt. In den Untersuchungen wurde die Praktikabilität dieser neuen Analyseverfahren im Routineeinsatz unter Standardbedingungen getestet und bestätigt. Die innovative Selektionsmethode der Annexin V-MACS Separation basiert auf der Bindung von Annexin V-MicroBeads an apoptotische Spermien, womit eine Subpopulation reifer, motiler und vitaler Spermien mit inaktivierter Apoptosesignalkaskade gezielt angereichert wird. Das Konzept wurde zudem erfolgreich auf ein (Glaswoll-) Festphasen-Filtersystem ohne frei schwimmende Microbeads übertragen. Dadurch gelang die Minimierung eines potentiellen Transmissionsrisikos der Microbeads bei der Anwendung im Rahmen der künstlichen Befruchtung. Den hohen Stellenwert dieser Verfahren belegen die Ergebnisse zweier in-vitro Modelle, an denen erstmalig gezeigt werden konnte, dass durch die Selektion von Spermien mit inaktivierter Apoptose-Signalkaskade höhere Fertilisationsraten erreichbar sind.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Development of bovine embryos produced by intracytoplasmic sperm injection (ICSI) is low compared to fertilized embryos. Deficient oocyte activation, inappropriate sperm capacitation, and lack of sperm decondensation are thought to be the main constrains affecting ICSI success in cattle. In the present study, activation compounds were tested to establish an effective protocol for activation of bovine oocytes, and then used for oocyte activation after ICSI. The activation approach consisted of exposing in vitro matured oocytes to Ionomycin (ION) following by a specific CDK1 inhibitor (RO-3306), a specific PKC activator (OAG) or both RO+OAG. In the first experiment, the rate of activation (pronuclear (PN) formation), cleavage, development to the blastocyst stage, and number of cells per blastocyst were evaluated after oocyte treatment. The PN rates were higher (P≤0.01) in the groups activated with ION+RO (48.5 %) and ION+RO+OAG (65.6 %) compared to ION (12.3 %) and ION+OAG (9.2 %). There was no significant effect between the RO concentrations tested (5, 7.5 and 10 μM) on oocyte activation. The PN rate was significantly higher (P≤0.01) when oocytes were exposed to RO for 240 min (84.6 %) compared to 60 (53.6 %) and 120 min (60.0 %). However, there was no difference between groups when treatment with RO started at 0, 30 or 60 min after on ION exposure. Cleavage rate was higher in ION+RO (70.2 %) and ION+RO+OAG (62.4 %) groups compared to ION (11.8 %) and ION+OAG (22.8 %). Blastocyst rate was also higher in the ION+RO+OAG (24.1 %) group, but not statistically different between ION+RO (19.7 %) and ION+OAG (9.5 %) groups. There was no development to the blastocyst stage after treatment with ION alone. The average cell number in blastocysts was not statistically different among treatments. In the second experiment, the effect of activation with ION+RO (10 μM for 240 min) was tested after ICSI using control (ICSI-Cont) or treated by electroporation (ICSI-El) sperm. Most oocytes presented a well-developed female PN (66.4%). Male PN formation was higher (P≤0.05) in the ICSI-El (33.3%) compared to the ICSI-Cont (9.4%) group. In conclusion, this study revealed that the specific inhibition of CDK1 after ION treatment is an effective approach to activate bovine oocytes. Male pronuclear formation after ICSI is increased by sperm electroporation, but is lower than female pronuclear formation. This indicates that deficient sperm decondensation and male PN formation rather than deficient oocyte activation is likely the main problem to develop an effective protocol for bovine ICSI. / O desenvolvimento de embriões bovinos produzidos por injeção intracitoplasmática de espermatozóides (ICSI) é baixo em relação aos embriões fertilizados. Deficiência na ativação de oócitos, capacitação inadequada de espermatozóides e falta de descondensação de espermatozóides são os principais transtornos que afetam o sucesso de ICSI em bovinos. No presente trabalho, foram testados métodos de ativação com o intuito de estabelecer um protocolo efetivo para a ativação de oócitos bovinos após a ICSI. Para isso, um inibidor específico de CDK1 (RO-3306) e um ativador específico de PKC (OAG) foram utilizados após a incubação com Ionomicina (ION) para avaliar a retomada da meiose em oócitos bovinos. Além disso, a incubação em meio Fert durante 6 h e a eletroporação (El) de espermatozoides previamente a ICSI foram testadas para verificar o efeito sobre a descondensação do espermatozoide e formação do pró-núcleo (PN) masculino. Inicialmente oócitos bovinos foram incubados, com diferentes concentrações e períodos de exposição aos tratamentos. Foram avaliados conforme a taxa de ativação oocitária (formação de PN), clivagem, desenvolvimento a blastocisto e número de células nos embriões que se desenvolveram a blastocisto. As taxas de PN foram maiores (P≤0,01) nos grupos ativados com ION+RO (48,5 %) e ION+RO+OAG (65,6 %) comparado com ION (12,3 %) e ION+OAG (9,2 %). Não houve efeito significativo entre as concentrações 5,0, 7,5 e 10,0 μM de RO sobre a taxa de ativação. A taxa de ativação foi significativamente maior (P≤0,01) em oócitos tratados com RO por 240 min (84,6 %) comparado a 60 (53,6 %) e 120 min (60,0%). No entanto, não houve diferença significativa na taxa de ativação quando o tratamento com RO foi iniciado a 0, 30 ou 60 minutos após a incubação com ION. A taxa de clivagem foi inferior nos grupos ION (11,8 %) e ION+OAG (22,8 %) comparada aos grupos ION+RO (70,2 %) e ION+RO+OAG (62,4 %). A taxa de blastocistos também foi maior no grupo ION+RO+OAG (24,1 %), mas não houve diferença estatística entre os grupos ION+RO (19,7 %) e ION+OAG (9,5 %). Não houve desenvolvimento a blastocisto quando os oócitos foram tratados somente com ION. Não foi detectada diferença estatística entre os tratamentos sobre o número médio de células por blastocistos. No segundo experimento, espermatozoides não tratados (ICSI-Cont) ou tratados por electroporação (ICSI-El) foram microinjetados em oócitos, os quais foram ativados com ION+RO por 240 min e fixados cerca de 15 h após a injeção para determinar a taxa de formação de PNs masculino e feminino (2PN). A maioria dos oócitos apresentaram o PN feminino bem desenvolvido (66,4 %). A formação de 2PN (masculino e feminino) foi maior no grupo ICSI-El (33,3 %) comparado ao grupo ICSI-Cont (9,4 %). Em conclusão, esse estudo demonstrou que a inibição específica da CDK1 após o tratamento com ION promove a ativação de oócitos bovinos. O tratamento de espermatozoides com eletroporação melhora a formação do PN masculino após ICSI, mas a taxa é inferior a formação do PN feminino. Esses resultados indicam que a deficiente descondensação do espermatozoide é o principal limitante para estabelecer um protocolo eficaz para ICSI em bovinos.

Introdução: Apesar de controverso, questiona-se um papel deletério da endometriose nos resultados de procedimentos de reprodução assistida, o que pode estar relacionado ao comprometimento da qualidade oocitária. Para que o oócito maduro esteja preparado para a fertilização, é necessário que o fuso meiótico mantenha a sua integridade e funcionabilidade. Objetivos: Comparar a presença e localização do fuso meiótico e o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis sem e com endometriose. Comparar os resultados de Injeção Intracitoplasmática de espermatozóides (ICSI) entre os oócitos em telófase I e metáfase II, e entre aqueles com e sem fuso celular visível, nos grupos analisados. Metodologia: Estudo prospectivo e controlado com pacientes inférteis, submetidas à estimulação ovariana para realização de ICSI, selecionadas consecutivamente e divididas em dois grupos: Controle (fator tubário e/ou masculino) e Endometriose (subdividido em endometriose mínima e leve I/II versus moderada e severa III/IV). Os oócitos com extrusão do primeiro CP foram avaliados pela microscopia de polarização imediatamente antes da realização da ICSI e caracterizados quanto à presença/localização do fuso celular em relação ao primeiro CP e ao estágio de maturação nuclear (telófase I ou metáfase II). Foram analisados as taxas de fertilização, clivagem, número de embriões de boa qualidade no segundo (D2) e terceiro (D3) dia de desenvolvimento oriundos dos oócitos em telófase I versus metáfase II, e metáfase II com fuso visível versus sem fuso visível, nos grupos controle, endometriose, endometriose I/II e endometriose III/IV. Resultados: Foram analisados 441 oócitos, sendo 254 do grupo controle e 187 do grupo endometriose (115 do grupo endometriose I/II e 72 do grupo endometriose III/IV). Não observamos diferença significativa entre a percentagem de oócitos em metáfase II com fuso celular visível e não visível (88,6%, 91,3%, 88,2%, respectivamente, nos grupos controle, endometriose I/II e endometriose III/IV) e entre a percentagem de oócitos com fuso celular nas diferentes localizações nos grupos avaliados. Entre os oócitos aparentemente maduros, observamos um aumento significativo de oócitos em telófase I no grupo endometriose III/IV (5,6%) quando comparado ao grupo endometriose I/II (0%). Observamos uma tendência a menores taxas de fertilização dos oócitos injetados em telófase I quando comparados aos em metáfase II, nos grupos controle (p=0,08), endometriose (p=0,05) e endometriose III/IV (p=0,09). Comparando-se os oócitos com e sem fuso celular visível, não observamos diferença significativa nos resultados de ICSI entre os grupos analisados. Conclusão: Não observamos diferença significativa entre os grupos analisados quanto à visualização e localização do fuso celular em oócitos maturados in vivo com o primeiro CP visível. Todavia, observamos um aumento significativo de oócitos em telófase I nas portadoras de endometriose moderada e severa, sugerindo um retardo ou comprometimento na conclusão da meiose I. Considerando que os oócitos injetados em telófase I apresentam piores taxas de fertilização do que os injetados em metáfase II, este achado poderia justificar o comprometimento dos resultados de reprodução assistida em mulheres inférteis com endometriose moderada e severa, além de ser utilizado com ferramenta prognóstica pós-ICSI. / Introduction: Although it has been a controversial issue for decades, a deleterious role of endometriosis on assisted reproductive techniques (ART) outcomes is questioned, which may be related to oocyte quality. For a mature oocyte be prepared for fertilization is necessary that the meiotic spindle keeps its integrity and its function. Objectives: To compare the presence and localization of the meiotic spindle and the oocyte nuclear maturation with the visible first polar body of infertile patients with and without endometriosis. To compare ICSI outcomes between oocytes on telophase I and metaphase II, and the ones with and without visible meiotic spindle, on those two groups. Methodology: A prospective and controlled study with infertile patients who underwent ovarian stimulation for purposes of ICSI, selected consecutively and divided into two groups: control (tubal and/or male factor) and endometriosis (subdivided in minimum and mild stage I/II versus moderate and severe stage III/IV). The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged using a polarization microscopy immediately before ICSI and characterized according to the presence and localization of meiotic spindle and its relation to the first polar body and the nuclear maturation stage (telophase I and metaphase II). We have analyzed the fertilization rates, clivage, number of good quality embryos on the second (D2) and third (D3) day of development from oocytes on telophase I versus the ones on metaphase II, and metaphase II visible spindle versus the non-visible ones, on the control groups, endometriosis, endometriosis stage I/II and endometriosis stage III/IV. Results: A total of 441 oocytes were analyzed, 254 oocytes form the control group and 187 from the endometriosis one (115 from endometriosis stage I/II and 72 from endometriosis stage III/IV). No significant differences between the percentage of metaphase II with visible and non-visible meiotic spindle were found (88,6%, 91,3%, and 88,2%, in the control, endometriosis I/II and endometriosis III/IV groups, respectively). Among the apparently matured oocytes, we have observed a significant increase of oocytes on telophase I on the endometriosis III/IV group (5,6%) when compared with the endometriosis I/II group (0%). We have observed a tendency to fewer fertilization rates from the injected oocytes on telophase I when compared with the ones on metaphase II, on the control group (p=0,08), endometriosis (p=0,05) and endometriosis III/IV group (p=0,09). When we compared oocytes with and without visible meiotic spindle, we found no significant difference on ICSI outcomes among the studied groups. Conclusions: We have found no significant difference among the studied groups regarding the visualization and localization of the meiotic spindle from in vivo matured oocytes with a visible first polar body. However, we have observed a significant increase on the number of oocytes on telophase I from patients with moderate and severe endometriosis, suggesting a delay or an impairment in the completion of meiosis I. Since the injected oocytes on telophase I present a worse fertilization rates than the ones injected on metaphase II, this finding could explain the impairment on the outcomes of ART in infertile women with moderate and severe endometriosis, besides it could be used as a prognosis tool after ICSI procedures.

Objetivos: Avaliar o fuso meiótico e a distribuição cromossômica de oócitos maturados in vitro obtidos de ciclos estimulados de mulheres inférteis com Síndrome dos Ovários Policísticos (SOP) e fatores masculino e/ou tubário de infertilidade. Métodos: Vinte e seis pacientes inférteis com SOP e 48 pacientes com fator tubário e/ou masculino de infertilidade, submetidas a ciclos estimulados para captação oocitária para injeção intracitoplasmática de espermatozóide, foram selecionadas prospectiva e consecutivamente e divididas em grupos de estudo e controle, respectivamente. Oócitos imaturos (34 e 56 oócitos) foram obtidos de 13 e 27 pacientes, respectivamente, dos grupos SOP e controle, sendo submetidos à maturação in vitro (MIV), respectivamente, por 19 horas ± 1 hora (VG) e 4 horas ± 30 minutos (MI), conforme curva de MIV previamente realizada no presente serviço. Oócitos em metáfase II (MII) após MIV, foram fixados, submetidos a imunocoloração e microscopia de fluorescência para avaliação morfológica do fuso e da distribuição cromossômica. Resultados: Não observamos diferença significativa nas taxas de MIV entre os dois grupos avaliados (50% e 42,8%, respectivamente, para os grupos SOP e controle). Na análise por microscopia de imunofluorescência, detectaram-se 3 e 2 oócitos, respectivamente, no grupo de estudo e no grupo controle, em estágio de Telófase I e 3 oócitos ativados partenogeneticamente no grupo controle. Ocorreu a impossibilidade de análise de 4 oócitos do grupo controle em virtude de dificuldades técnicas durante o processo de imunocoloração. Não houve diferença significativa nas proporções de anomalias meióticas entre os grupos SOP e controle (57,1 e 46,7%, respectivamente). Conclusões: Os dados preliminares do presente estudo, apesar de não demonstrarem aumento significativo na incidência de anomalias meióticas nas portadoras de SOP, sugerem uma tendência a maior ocorrência de anomalias meióticas nos oócitos deste grupo de pacientes, quando comparados aos de portadoras de fator masculino e/ou tubário de infertilidade, o que deverá ser mais bem avaliado em estudos com maiores casuísticas. Estes achados têm potencial clínico para apontar uma possível explicação para as controversas menores taxas de fertilização observadas em pacientes com SOP submetidas às Técnicas de Reprodução Assistida. / Objectives: To evaluate the meiotic spindle and the chromosome distribution of in vitro matured oocytes obtained during stimulated cycles from infertile women with Polycystic Ovary Syndrome (PCOS) and with male factor and/or tubal infertility. Methods: Twenty six infertile patients with PCOS and 48 patients with infertility due to tubal and/or male factor, submitted to stimulated cycles for oocyte retrieval for intracytoplasmic sperm injection, were selected prospectively and consecutively and respectively assigned to the study group and the control group. Imature oocytes (34 and 56 oocytes) were obtained from 13 and 27 patients, respectively, of PCOS and control groups, and submitted to in vitro maturation (IVM) for 19 hours ± 1 hour (GV) and 4 hours ± 30 minutes (MI) according to the IVM curve previously constructed in the present service. After IVM, oocytes in metaphase II (MII) were fixed and submitted to immunostaining and fluorescence microscopy for morphological evaluation of the spindle and of chromosome distribution. Results: IVM rates were similar between the two analyzed groups (50% e 42.8%, respectively, in PCOS e control groups). By immunofluorescence analysis, there were 3 and 2 oocytes, respectively, in PCOS e control groups, in telophase I stage, and 3 parthenogenetic activated oocytes in control group. Because of technical difficulties during the execution of the immunofluorescence protocol, 4 oocytes from the control group could not be analyzed. The difference in the proportions of meiotic anomalies between the two groups was not statistically significant (57.1 e 46.7%, respectively, in PCOS e control groups). Conclusions: The present preliminary data, although not showing a significant increase in the incidence of meiotic anomalies in women with PCOS, suggest a tendency to a higher occurrence of meiotic anomalies in the oocytes of this group of patients compared to women with male and/or tubal infertility, a fact to be better evaluated in studies on larger patient series. The present findings have the clinical potential to provide a possible explanation for the controversial lower fertilization rates observed in patients with PCOS submitted to Assisted Reproduction Techniques