Made available in DSpace on 2015-03-03T11:52:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-02Bitstream added on 2015-03-03T12:06:28Z : No. of bitstreams: 1 000806927.pdf: 1884256 bytes, checksum: 34e30cf4b909e11722195260f1d006ce (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A produção de embrião in vitro (PIV) tem sido uma grande alternativa para os programas de preservação de felídeos selvagens ameaçados de extinção. No entanto, a PIV necessariamente depende de outras técnicas de reprodução assistida como a maturação in vitro (MIV), fertilização in vitro (FIV), transferência de embrião (TE), e criopreservação de gametas. Essas técnicas de reprodução assistida já foram transferidas com sucesso do gato doméstico para seus parentes selvagens. Já o uso de sêmen liofilizado também resultou em filhotes vivos de diversas espécies de produção, mas em gatos domésticos isso ainda não ocorreu. O objetivo do presente estudo foi verificar a fragmentação do DNA do sêmen liofilizado e a viabilidade de seu uso através da utilização da ICSI para a PIV de embriões de gatos domésticos. Foram colhidas amostras de 3 gatos normospérmicos que foram utilizadas a fresco, congeladas/descongeladas e liofilizadas. Foram realizados testes para avaliação de integridade de DNA (Laranja de Acridina Convencional, Ensaio do Cometa Alcalino, Laranja de Acridina na citometria de fluxo, e TUNEL). O método de TUNEL mostrou ter maior sonsibilidade para detecção de fragmentação de DNA e detectou grau elevado de lesão de DNA do sêmen liofilizado em comparação com o sêmen fresco e congelado/descongelado dos mesmos doadores. Não foi possível produzir embrião de gato através da técnica de ICSI utilizando sêmen que foi liofilizado em meio SOF / The in vitro production of embryos (IVP) has been a good alternative for conservational programmes of wild felids threatened by extinction. However, IVP depends upon several other assisted reproduction techniques such as in vitro maturation (IVM), in vitro fertilization (IVF), embryo transfer (ET), gamete cryopreservation. These techniques were already successfully employed in wild felines. Freeze-dried sperm has also been used to produce live offspring of several species, but in domestic cat this has not been reported yet. Hence, the objective of the present study was to verify the sperm DNA fragmentation after the freeze-drying process as well as the feasibility of using freeze-dried sperm together with ICSI to produce cat IVP embryos. Fresh, frozen/thawed, freeze-dried sperm samples of 3 normospermic cats were used in this experiment. Sperm DNA integrity assessments were performed (Conventional Acridine Orange, Alkaline Comet Assay, Acridine Orange using flow cytometry, and TUNEL). TUNEL assessment showed higher sensitivity to detect DNA fragmentation and it detected higher DNA fragmentation in freeze-dried sperm when compared to frozen/thawes and fresh semen from the same donors. It was not possible to produce IVP embryos making use of freezedried sperm that used SOF as medium and ICSI

Orientador: Maria Denise Lopes / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: Jussara Maria Tebet / Banca: Maria Isabel Mello Martins / Banca: Camila de Paula Freitas Dell'Aqua / Resumo: A produção de embrião in vitro (PIV) tem sido uma grande alternativa para os programas de preservação de felídeos selvagens ameaçados de extinção. No entanto, a PIV necessariamente depende de outras técnicas de reprodução assistida como a maturação in vitro (MIV), fertilização in vitro (FIV), transferência de embrião (TE), e criopreservação de gametas. Essas técnicas de reprodução assistida já foram transferidas com sucesso do gato doméstico para seus parentes selvagens. Já o uso de sêmen liofilizado também resultou em filhotes vivos de diversas espécies de produção, mas em gatos domésticos isso ainda não ocorreu. O objetivo do presente estudo foi verificar a fragmentação do DNA do sêmen liofilizado e a viabilidade de seu uso através da utilização da ICSI para a PIV de embriões de gatos domésticos. Foram colhidas amostras de 3 gatos normospérmicos que foram utilizadas a fresco, congeladas/descongeladas e liofilizadas. Foram realizados testes para avaliação de integridade de DNA (Laranja de Acridina Convencional, Ensaio do Cometa Alcalino, Laranja de Acridina na citometria de fluxo, e TUNEL). O método de TUNEL mostrou ter maior sonsibilidade para detecção de fragmentação de DNA e detectou grau elevado de lesão de DNA do sêmen liofilizado em comparação com o sêmen fresco e congelado/descongelado dos mesmos doadores. Não foi possível produzir embrião de gato através da técnica de ICSI utilizando sêmen que foi liofilizado em meio SOF / Abstract: The in vitro production of embryos (IVP) has been a good alternative for conservational programmes of wild felids threatened by extinction. However, IVP depends upon several other assisted reproduction techniques such as in vitro maturation (IVM), in vitro fertilization (IVF), embryo transfer (ET), gamete cryopreservation. These techniques were already successfully employed in wild felines. Freeze-dried sperm has also been used to produce live offspring of several species, but in domestic cat this has not been reported yet. Hence, the objective of the present study was to verify the sperm DNA fragmentation after the freeze-drying process as well as the feasibility of using freeze-dried sperm together with ICSI to produce cat IVP embryos. Fresh, frozen/thawed, freeze-dried sperm samples of 3 normospermic cats were used in this experiment. Sperm DNA integrity assessments were performed (Conventional Acridine Orange, Alkaline Comet Assay, Acridine Orange using flow cytometry, and TUNEL). TUNEL assessment showed higher sensitivity to detect DNA fragmentation and it detected higher DNA fragmentation in freeze-dried sperm when compared to frozen/thawes and fresh semen from the same donors. It was not possible to produce IVP embryos making use of freezedried sperm that used SOF as medium and ICSI / Doutor

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-01Bitstream added on 2014-06-13T19:39:22Z : No. of bitstreams: 1 cota_amm_me_botfm.pdf: 542817 bytes, checksum: 25407ae609f35711de3e74eccfa2d0a3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na reprodução assistida, a seleção de gametas com o objetivo de alcançar melhores resultados clínicos é uma tarefa crucial dos embriologistas. A qualidade do oócito é um fator chave na fertilidade feminina, refletindo o potencial intrínseco de desenvolvimento do gameta, além de ter um papel crucial não só na fecundação, mas também no desenvolvimento embrionário subsequente. Após a desnudação, consegue-se definir a maturidade oocitária, com a identificação do primeiro corpúsculo polar, além de permitir a avaliação da morfologia oocitária, analisando as características da zona pelúcida, do espaço perivitelino e do citoplasma. Os dismorfismos oocitários são classificados em 2 tipos: citoplasmáticos, que incluem a presença de granulações e/ou de inclusões citoplasmáticas (vacúolos, corpos refrativos, agregados do retículo endoplasmático) e extracitoplasmáticos (alterações na forma do oócito, alterações na zona pelúcida, no espaço perivitelino e alterações do corpúsculo polar). Essas variações na morfologia oocitária podem ocorrer devido a fatores como idade da mulher, problemas genéticos e alterações no ambiente hormonal a que o oócito é exposto com a hiperestimulação ovariana. A classificação da morfologia oocitária, bem como sua correlação com o desenvolvimento embrionário e taxa de gravidez são bastante controversas na literatura. Vários estudos não demonstram nenhuma associação entre os dismorfismos oocitários e os resultados da fertilização in vitro, enquanto outros relatam uma associação entre a morfologia oocitária e desenvolvimento embrionário. Essas diferenças nos resultados podem ser explicadas devido a utilização de diferentes critérios morfológicos e devido... / The selection of developmentally competent human gametes may increase the efficiency of assisted reproduction. Spermatozoa and oocytes are usually assessed according to morphologic criteria. Oocyte morphology can be affected by the age of the female, genetic aspects, and factors related to controlled ovarian stimulation. However, there is a lack of evidence in the literature concerning the effect of gonadotropin-releasing hormone (GnRH) analogues, either agonists or antagonists, on oocyte morphology. The aim of this randomized study was to investigate if the prevalence of oocyte dysmorphism is influenced by the type of pituitary suppression used in ovarian stimulation. A total of 64 patients at the first intracytoplasmic sperm injection (ICSI) cycle, were prospectively randomized to receive treatment with either a GnRH agonist with a long-term protocol (n: 32) or a GnRH antagonist with a multi-dose protocol (n: 32). Before being subjected to ICSI, the oocytes at metaphase II from both groups were morphologically analyzed under an inverted light microscope at a 400x magnification. The oocytes were classified as follows: normal or with cytoplasmic dysmorphism, extracytoplasmic dysmorphism, or both. The resulting measure was the detection of dysmorphic oocytes among the total number of oocytes analyzed. Out of a total of 681 oocytes, 189 (27.8%) were morphologically normal, 220 (32.3%) showed cytoplasmic dysmorphism, 124 (18.2%) showed extracytoplasmic alterations, and 148 (21.7%) exhibited both types of dysmorphisms. No significant difference was observed in oocyte dysmorphisms between the agonist- and antagonisttreated groups (P>0.05). Analysis for each dysmorphism revealed that the most common conditions were alterations in polar body shape (31.3%) and presence of diffuse cytoplasmic granulations (22.8%), refractile bodies (18.5%) and central cytoplasmic... (Complete abstract click access electronic below)

Abstract The effectiveness of intrauterine insemination (IUI) combined with controlled ovarian hyperstimulation (COH) in the treatment of subfertility was investigated in the present study. For this purpose the prognostic factors associated with success of clomiphene citrate (CC)/human menopausal gonadotrophin (HMG)/IUI were identified in 811 treatment cycles. Furthermore, a long gonadotrophin-releasing hormone agonist (GnRHa)/HMG stimulation protocol was compared with a standard CC/HMG protocol. In addition, the usefulness of alternative insemination techniques including fallopian tube sperm perfusion (FSP) and intrafollicular insemination (IFI) was investigated. Finally, the obstetric and perinatal outcome of pregnancies after COH/IUI was examined and compared with those of matched spontaneous and in vitro fertilization(IVF) pregnancies. Female age, duration of infertility, aetiology of infertility, number of large preovulatory follicles and number of the treatment cycle were predictive as regards pregnancy after CC/HMG/IUI. The highest pregnancy rate (PR) was obtained in women of < 40 years of age with infertility duration ≤ 6 years, who did not suffer from endometriosis. A multifollicular ovarian response to CC/HMG resulted in better treatment success than a monofollicular response, indicating the necessity of COH combined with IUI. A significantly higher PR was achieved in the first treatment cycles compared with the others, and 97% of the pregnancies were obtained in the first four treatment cycles. The PR per cycle did not differ significantly between a long GnRHa/HMG and a standard CC/HMG protocol, but the average medication expense of GnRHa/HMG stimulation was four times the cost of CC/HMG stimulation. Therefore, the routine use of a long GnRHa/HMG protocol in IUI treatment remains questionable. The FSP procedure was easy to perform by using a paediatric Foley catheter. The success rate in couples with either FSP or standard IUI did not differ significantly, although there was a trend towards a lower PR in the FSP group. The FSP technique should not replace the simpler and less time-consuming IUI technique in routine use. The IFI technique was also simple to perform and convenient for patients. However, only one normal singleton intrauterine pregnancy resulted in 50 IFI-treated women, indicating that IFI is inefficacious for treating subfertility. The IUI parturients differed from average Finnish parturients in respect to higher maternal age, more frequent primiparity and a higher incidence of multiple pregnancies. The use of antenatal care services was significantly lower in IUI singleton pregnancies compared with IVF singletons, although there were no more complications in IVF pregnancies. The hospitalization and Caesarean section rates were generally high in all pregnancies. The mean birthweight of IUI singletons was significantly lower than that in spontaneous pregnancies, but comparable to that in IVF pregnancies. However, the incidence of preterm birth, low birth weight and other variables describing the outcome of infants were similar in IUI, IVF and spontaneous pregnancies. In summary, the IUI procedure itself does not seem to affect adversely the obstetric and perinatal outcome of pregnancy, and patient characteristics and multiplicity may be more important in this respect.

Orientador: Joao Batista Alcantara Oliveira / Coorientador: Claudia Guilhermino Petersen / Banca: Mario Cavagna / Banca: Anice Maria Vieira de Camargo Martins / Resumo: Na reprodução assistida, a seleção de gametas com o objetivo de alcançar melhores resultados clínicos é uma tarefa crucial dos embriologistas. A qualidade do oócito é um fator chave na fertilidade feminina, refletindo o potencial intrínseco de desenvolvimento do gameta, além de ter um papel crucial não só na fecundação, mas também no desenvolvimento embrionário subsequente. Após a desnudação, consegue-se definir a maturidade oocitária, com a identificação do primeiro corpúsculo polar, além de permitir a avaliação da morfologia oocitária, analisando as características da zona pelúcida, do espaço perivitelino e do citoplasma. Os dismorfismos oocitários são classificados em 2 tipos: citoplasmáticos, que incluem a presença de granulações e/ou de inclusões citoplasmáticas (vacúolos, corpos refrativos, agregados do retículo endoplasmático) e extracitoplasmáticos (alterações na forma do oócito, alterações na zona pelúcida, no espaço perivitelino e alterações do corpúsculo polar). Essas variações na morfologia oocitária podem ocorrer devido a fatores como idade da mulher, problemas genéticos e alterações no ambiente hormonal a que o oócito é exposto com a hiperestimulação ovariana. A classificação da morfologia oocitária, bem como sua correlação com o desenvolvimento embrionário e taxa de gravidez são bastante controversas na literatura. Vários estudos não demonstram nenhuma associação entre os dismorfismos oocitários e os resultados da fertilização in vitro, enquanto outros relatam uma associação entre a morfologia oocitária e desenvolvimento embrionário. Essas diferenças nos resultados podem ser explicadas devido a utilização de diferentes critérios morfológicos e devido... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The selection of developmentally competent human gametes may increase the efficiency of assisted reproduction. Spermatozoa and oocytes are usually assessed according to morphologic criteria. Oocyte morphology can be affected by the age of the female, genetic aspects, and factors related to controlled ovarian stimulation. However, there is a lack of evidence in the literature concerning the effect of gonadotropin-releasing hormone (GnRH) analogues, either agonists or antagonists, on oocyte morphology. The aim of this randomized study was to investigate if the prevalence of oocyte dysmorphism is influenced by the type of pituitary suppression used in ovarian stimulation. A total of 64 patients at the first intracytoplasmic sperm injection (ICSI) cycle, were prospectively randomized to receive treatment with either a GnRH agonist with a long-term protocol (n: 32) or a GnRH antagonist with a multi-dose protocol (n: 32). Before being subjected to ICSI, the oocytes at metaphase II from both groups were morphologically analyzed under an inverted light microscope at a 400x magnification. The oocytes were classified as follows: normal or with cytoplasmic dysmorphism, extracytoplasmic dysmorphism, or both. The resulting measure was the detection of dysmorphic oocytes among the total number of oocytes analyzed. Out of a total of 681 oocytes, 189 (27.8%) were morphologically normal, 220 (32.3%) showed cytoplasmic dysmorphism, 124 (18.2%) showed extracytoplasmic alterations, and 148 (21.7%) exhibited both types of dysmorphisms. No significant difference was observed in oocyte dysmorphisms between the agonist- and antagonisttreated groups (P>0.05). Analysis for each dysmorphism revealed that the most common conditions were alterations in polar body shape (31.3%) and presence of diffuse cytoplasmic granulations (22.8%), refractile bodies (18.5%) and central cytoplasmic... (Complete abstract click access electronic below) / Mestre

Genetic disorders like Cystic Fibrosis (CF) and X-linked Diseases (XLD) are inherited by offspring from parents who are healthy carriers of the autosomal recessive or allosomal genes. About 10-million Americans are healthy carriers of a mutant cysticfibrosis gene (predominantly F508del) and about 4% of newborns are at risk of being born with an X-linked disease. The current clinically approved mitigation plan for preventing genetic disorders in newborns from “at-risk couples” is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M). PGT-M involves an invasive microsurgical procedure that requires the removal of cells from 3-5day old embryos. To minimize this invasiveness, we proposed a less invasive approach to prevent genetic disorders in newborns by genotypically sorting sperm cells which may be used for fertilization events (IUI/IVF/ICSI) with specially characterized antigens on the sperm surface membrane. For the disease models being adopted in our study – CF and XLD; we utilized certain monoclonal antibodies (mab) to target the H-Y male antigen and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein which are both selectively expressed on the sperm surface. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

This study details the Medically Assisted Procreation regulations in thirty-five nation-states, and explores the influence of national identity, social cultural and demographic differences on these regulations. Detailed data were gathered from ministries of health, offices of prime ministers, embassy staff, and others on regulations for each nation. These data were used to categorize the nations in regard to MAP legislation status and regulatory policy regarding marital or age restrictions; posthumous conception; sperm, ovum, or embryo donation, surrogacy; and policy on handling donors. Possible associations between national identity, social cultural, and demographic data for each nation and their regulations were explained. The thirty-five nations were treated as a population with common geographical and political ties. PRE methods, and eta coefficients were used to assess the associations. Sixteen nations have adopted MAP legislation, eight nations have either alternative regulatory guidelines or partial structures, four nations have legislation pending and possibly some laws, and seven nations are unregulated. Based upon statistical analysis, language group emerges as an important indicator for differences in MAP regulations. For example knowing a nation's language group enabled percent improved prediction of that nation's regulatory handling of embryo donation. The percent GDP spent on health care was found to have a substantial or moderate association with most regulations. The findings of this study indicate that the cultural roots associated with national identity as well as economic circumstances such as health care budgets impact the policy making process responsible for the regulation of MAP in Europe. Among other mediating circumstances, MAP related family law cases brought to the European Court of Human Rights create an accumulation of judge-made law, which help create a common European standard. This study of the European region provides a baseline for further research and a reference for cross cultural comparisons.

There have been leaps in both fields of epigenetics and reproductive technology. This has culminated in the production of bi-maternal mouse offspring through a few studies utilizing direct gene mutations as functional-imprints. While these genetic interventions result in positive results, it has yet to be described, in full, what mechanisms underlie the epigenetic barriers to human gynogenesis. Between maternal and paternal imprints, differences in methylation patterns of CpG island promoters, non-coding regions, microsatellites, transposons, and histones result in differences in higher order chromatin structure, which ultimately impacts expression of certain genes. While the necessary components of a minimal paternal epigenetic program are described, programming this imprint onto m2, a hypothetical, experimentally-produced maternal genome with a paternal epigenome is still not elucidated. Sequential timing of imprint acquisition and maintenance and the numerous regulatory factors associated with them illuminate how difficult potential assisted reproductive epigenetic interventions will be. Other processes like histone-protamine exchange which also plays a crucial factor in structural regulation of imprints, as well as signaling through and after fertilization, pose logistical problems to creating a gynogenote through epigenetic means. Lastly, ethics surrounding the introduction of dangerous mutations and epialleles into the human population add another wall of caution and hesitance in exploring human gynogenesis as a reproductive technology.

No description available.

No description available.