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OBJECTIVE: To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). METHODS: Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. RESULTS: The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. CONCLUSIONS: PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.

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INTRODUÇÃO: A oncofertilidade tem o desafio de buscar estratégias para preservar a função reprodutiva. Este estudo explorou duas possibilidades como implementação para as técnicas de preservação da fertilidade feminina e masculina. OBJETIVO: Analisar a eficiência do cultivo de folículos pré-antrais de camundongos suplementado com lisado de plaquetas humanas e desenvolver um protótipo para criopreservação de sêmen humano. MATERIAIS E MÉTODOS: Os folículos pré-antrais foram isolados mecanicamente de ovários de fêmeas de camundongos e foram cultivados individualmente em sistema entre camadas de óleo mineral. Os folículos foram cultivados divididos em 4 grupos, sendo um controle, sem o uso do lisado de plaquetas e três grupos com diferentes concentrações de lisado de plaquetas humanas (PLTMax®). Foram avaliadas a sobrevivência celular, desenvolvimento folicular e características oocitárias. Para o segundo estudo foi desenvolvido e impresso em 3D com filamentos de acrilonitrilo butadieno-estireno (ABS) um protótipo que suporte 10 palhetas com amostras seminais no vapor de nitrogênio líquido (N2L), etapa essencial para criopreservação de sêmen humano. Para os testes foram utilizadas 40 amostras seminais. A temperatura ambiente e no interior das palhetas de envase das amostras foram medidas e estabelecida a curva de resfriamento. Os parâmetros de motilidade, vitalidade e fragmentação do DNA espermático foram avaliados antes do congelamento e após o descongelamento. Foram realizados dois testes, um de posicionamento das palhetas e outro comparativo entre o protótipo e um dispositivo com suporte em poliestireno expandido (EPS). RESULTADOS: O cultivo de 11 dias induziu um aumento no tamanho folicular em todas as condições, sendo maior no grupo controle, seguido do grupo com 10% de PLTMax®, mas com diferença significativa (p<0,001). O grupo controle apresentou maior número de oócitos intactos (>50%) em relação aos demais (<35%). Todos os 4 grupos apresentaram taxas de vitalidade celular acima de 70%. Quanto aos testes com o protótipo em ABS foi verificado que as curvas de refrigeração foram notavelmente reproduzíveis. O material do protótipo resistiu a inúmeros mergulhos (>300) no N2L, sem demonstrar danos ao material. Diferenças significativas (p<0,001) foram observadas para a taxa de recuperação média da motilidade e vitalidade espermática em relação aos dados da amostra 2 fresca em ambos os testes. A motilidade, a vitalidade e a fragmentação do DNA espermático antes do congelamento e após o descongelamento não mostraram diferenças em relação a posição das palhetas. Também não houve diferença quanto ao índice de fragmentação verificada das amostras criopreservadas com uso do protótipo em ABS e o suporte em EPS, mesmo após o cultivo, após 24 horas de cultivo. Contudo, houve diferença em relação a amostra fresca (p<00,1). Quanto a recuperação das taxas de motilidade e vitalidade não houve diferença entre o ABS e EPS após o descongelamento e 24 horas de cultivo. CONCLUSÃO: O PLTMax®, embora tenha apresentado menor desempenho que o HSA, é um candidato de suplementação para o cultivo de folículos pré-antrais que merece ser mais explorado. O protótipo em ABS demonstrou resistência, praticidade e segurança para criopreservação seminal de forma reprodutível e eficiente.

INTRODUCTION: Oncofertility has the challenge of seeking strategies to preserve reproductive function. This study explored two possibilities as implementations for female and male fertility preservation techniques. PURPOSE: To analyze the efficiency of mouse preantral follicle culture supplemented with human platelet lysate and to develop a prototype for human semen cryopreservation. MATERIAL AND METHODS: Preantral follicles were mechanically isolated from female mouse ovaries and were individually cultured using a mineral oil interlayer system. The follicles were cultured divided into 4 groups, one control, without the use of platelet lysate and three groups with different concentrations of human platelet lysate (PLTMax®). Cell survival, follicular development and oocyte characteristics were evaluated. For the second study, a prototype was developed and printed in 3D with acrylonitrile butadiene styrene (ABS) filaments to support 10 straws with seminal samples in liquid nitrogen (N2L) vapor, an essential step for human semen cryopreservation. For the tests 40 seminal samples were used. Ambient and internal temperatures inside the sample straws were measured and the cooling curve was established. The parameters of motility, vitality and sperm DNA fragmentation were evaluated before freezing and after thawing. Two tests were performed, one for positioning the straws and the other comparing the prototype and a device with expanded polystyrene (EPS) support. RESULTS: The 11-day culture induced an increase in follicular size in all conditions, being higher in the control group followed by the group with 10% PLTMax®, but with significant difference (p<0.001). The control group presented a higher number of intact oocytes (>50%) compared to the others (<35%). All 4 groups presented cell vitality rates above 70%. As for the ABS prototype tests, it was verified that the cooling curves were remarkably reproducible. The prototype withstood numerous dips (>300) in N2L without showing damage to the material. Significant differences (p<0.001) were observed for the mean recovery rate of sperm motility and vitality compared to the fresh sample data in both tests. Motility, vitality and sperm DNA fragmentation before freezing and after thawing showed no differences with respect to the position of the straws. There was also no difference in the fragmentation index verified for samples cryopreserved using the ABS prototype and the EPS support, even after 24 hours of culture. However, there was a difference compared to the fresh 4 sample (p<00.1). As for the recovery of motility and vitality rates there was no difference between ABS and EPS after thawing and 24 hours of culture. CONCLUSION: PLTMax®, although it showed lower performance than HSA, is a supplementation candidate for preantral follicle culture that deserves further exploration. The ABS prototype demonstrated strength, practicality and safety for seminal cryopreservation in a reproducible and efficient manner.

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OBJECTIVE: To present the case of a man with normozoospermia and a high level of fragmented spermatozoa, which origin seems to be associated with long-term treatment with terbinafine hydrochloride. CASE DESCRIPTION: A 20-year-old male healthy patient, with no history of disease and addictions, used an antifungal (terbinafine hydrochloride) for one year to treat a toenail. During this treatment, he participated in a study to evaluate a method of sperm DNA fragmentation analysis. He had 99% fragmented sperm, primarily attributed to prolonged abstinence. The samples that were analyzed later indicated that the high fragmentation could be associated with the antifungal treatment and that with a 2-day abstinence and absence of treatment the fragmentation rate was again comparable with that of fertile men (15%). CONCLUSION: Terbinafine hydrochloride is likely to cause problems in male fertility, mainly affecting DNA sperm integrity. Further studies are needed to confirm this observation and to determine at what level of the genitourinary tract the alteration of DNA occurs.

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OBJECTIVE: To evaluate in vitro oocyte maturation rates in embryonic culture medium after induction by hyperosmotic shock caused by exposure to vitrification solutions. METHODS: Bilateral oophorectomy was performed on 20 prepubescent female mice (Swiss). Immature (Prophase I) oocytes (N = 400) were obtained by ovarian dissection, divided into 4 groups, and transferred to culture dishes containing fertilization medium (Sydney IVF Fertilization Medium, Cook® Medical). The control group (CG) did not receive treatment, the test groups (G1, G2, G3) were treated with vitrification solution - 2 (VI-2: 14 M sucrose + ethylene glycol and dimethyl sulfoxide) for 30 seconds and subsequently: G1: 30 seconds in devitrification solution - 2 (DV-2: 0.5M sucrose); G2: 60 seconds DV-2; G3: 60 seconds DV-1(1M sucrose) and 180 seconds DV-2. All groups were cultivated for 24 hours in an incubator at 37ºC and 5% CO2 (Thermo model 3110). After this period, we checked their maturation status. RESULTS: Oocytes exposed to VI-2, DV-1 and DV-2 (G3) showed the highest rate of competence in resuming meiosis and reaching the MII stage; however, there was no statistically significant difference (G3 = 50.5% - 49/97; CG = 27.8% - 10/30). CONCLUSIONS: Oocyte exposure to vitrification solutions, in order to cause osmotic shock, did not interfere with the resumption of meiosis in mice oocytes.

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OBJECTIVE: To evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation. METHODS: Twenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed. RESULTS: The viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site. CONCLUSION: The vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

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OBJECTIVE: To evaluate the efficiency of ovarian tissue heterotopic autografting after vitrification in prepubertal rats. METHODS: Fragments of excised ovaries from prepubertal rats were used after assessing post-warming cellular viability, to determine the best vitrification protocol prior to retroauricular autografting. Pre-pubertal females (N=24) were castrated and divided into three group: Group 1 - fresh ovarian tissue transplantation; Group 2 - vitrified/warmed tissue transplantation; Group 3 - bilateral oophorectomy without transplantation. The ovarian fragments were exposed to solutions from the Ingamed® commercial kit, allocated in bacteriological loops and immersed in liquid nitrogen. Sixty days after transplantation, a vaginal mucus sample was collected for cytology tests, followed by sacrificing the animal, performing a cardiac puncture for collecting a blood sample to determine luteinizing hormone and estradiol levels, and excision of the transplanted fragment for histology tests. RESULTS: Vaginal cytology revealed that 87.5% of females from groups 1 and 2 had estrus while all females in Group 3 remained in diestrus. The mean LH value in groups 1 (0.08 mIU/mL) and 2 (0.34 mIU/mL) were statistically different from that of Group 3 (2.27 mIU/mL). E2 values did not differ between the groups. The histological analysis of Group 1 excised grafts versus those from Group 2 showed a higher percentage of primary follicles (62.5% vs. 12.5%), developing follicles (75% vs. 25%), corpus luteum (37.5% vs. 12.5%) and stromal region (100% vs. 87.5%). CONCLUSION: This study indicated that pre-pubertal ovarian tissue vitrification can be used to preserve fertility and to restore endocrine function in castrated rats.

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BACKGROUND: The present study evaluates the effects of energy drinks on the reproductive and biochemical parameters of adult male rats. METHODS: A total of 40 male rats (Wistar) were exposed to an energy drink mixed with the drinking water for a period of 120 days. The animals were divided into four groups and exposed to increasing therapeutic doses (DT) of an energy drink, based on allometric extrapolation, resulting in values (mL/day) per animal of 250 g: DT1 2.36 mL, DT3 7.47 mL, and DT6 14.16 mL. The control group (CTRL) consumed water only. During the treatment, the rats were assessed for signs of toxicity. After treatment, the animals were sacrificed and their organs were weighed. Sperm parameters (motility, concentration, and morphology) were evaluated. The biochemical markers alanine eamino transferase, aspartate amino transferase, alkaline phosphatase, lactic dehydrogenase, urea, creatinine, creatine phosphokinase, and creatine kinase MB fraction were measured, in addition to total cholesterol and testosterone. RESULTS: There was a significant decrease (p< 0.05) in the concentration of sperm in the treated groups (DT18.5 ± 0.7; DT3 7.2 ± 0.9; DT6 8.4 ± 0.9) compared to the control group (12.3 ± 1.2). No difference was observed with respect to relative weights of the animals'organs, water consumption, signs of toxicity, behavioral changes, biochemical markers, and sperm motility and morphology. CONCLUSION: The long-term consumption of energy drinks interferes negatively with sperm concentration, without affecting sperm motility and morphology or altering the hepatic, cardiac, or renal functions

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Tecnologias reprodutivas têm uma participação fundamental na pesquisa biomédica. Por meio delas,novos modelos animais são produzidos e preservados de forma segura. A produção de animais geneticamentemodificados envolve diretamente o uso de embriões, sua manipulação e transferência para fêmeas receptoras.Uma vez produzida uma nova linhagem geneticamente modificada, a melhor forma de preservá-la é por meio dacriopreservação de espermatozoides ou embriões. Uma nova tecnologia para manipulação e modificação dogenoma do camundongo foi desenvolvida nos últimos anos. O sistema CRISPR/Cas9 tornou a produção destesanimais mais rápida, efetiva e com um custo mais baixo que as técnicas tradicionais. Esta tecnologiaimpulsionará o projeto mundial de produção de animais mutantes para cada um dos cerca de 25 mil genespresentes no camundongo. Ainda permitirá a produção de modelos com mutação dupla ou tripla e em animaiscom fundo genético distintos. Esta apresentação abordará a importância das técnicas reprodutivas neste processofundamental da pesquisa biomédica.

Reproductive technologies have a fundamental role in biomedical research. Through them, new animal modelsare produced and preserved safely. The production of genetically modified animals directly involves the use ofembryos, their manipulation and transference to recipient females. Once a new genetically modified lineage hasbeen produced, the best way to preserve it is by cryopreservation of sperm or embryos. A new technology formanipulating and modifying the mouse genome has been developed in recent years. The CRISPR / Cas9 systemmade the production of these animals faster, more effective and at a lower cost than traditional techniques. Thistechnology will boost the worldwide mutant animal production project for each of the approximately 25,000genes present in the mouse. It will also allow the production of models with double or triple mutation and inanimals with different genetic backgrounds. This presentation will address the importance of reproductivetechniques in this fundamental process of biomedical research.

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Tecnologias reprodutivas têm uma participação fundamental na pesquisa biomédica. Por meio delas,novos modelos animais são produzidos e preservados de forma segura. A produção de animais geneticamentemodificados envolve diretamente o uso de embriões, sua manipulação e transferência para fêmeas receptoras.Uma vez produzida uma nova linhagem geneticamente modificada, a melhor forma de preservá-la é por meio dacriopreservação de espermatozoides ou embriões. Uma nova tecnologia para manipulação e modificação dogenoma do camundongo foi desenvolvida nos últimos anos. O sistema CRISPR/Cas9 tornou a produção destesanimais mais rápida, efetiva e com um custo mais baixo que as técnicas tradicionais. Esta tecnologiaimpulsionará o projeto mundial de produção de animais mutantes para cada um dos cerca de 25 mil genespresentes no camundongo. Ainda permitirá a produção de modelos com mutação dupla ou tripla e em animaiscom fundo genético distintos. Esta apresentação abordará a importância das técnicas reprodutivas neste processofundamental da pesquisa biomédica.(AU)

Reproductive technologies have a fundamental role in biomedical research. Through them, new animal modelsare produced and preserved safely. The production of genetically modified animals directly involves the use ofembryos, their manipulation and transference to recipient females. Once a new genetically modified lineage hasbeen produced, the best way to preserve it is by cryopreservation of sperm or embryos. A new technology formanipulating and modifying the mouse genome has been developed in recent years. The CRISPR / Cas9 systemmade the production of these animals faster, more effective and at a lower cost than traditional techniques. Thistechnology will boost the worldwide mutant animal production project for each of the approximately 25,000genes present in the mouse. It will also allow the production of models with double or triple mutation and inanimals with different genetic backgrounds. This presentation will address the importance of reproductivetechniques in this fundamental process of biomedical research.(AU)

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The objective of this study was to evaluate the the efficiency of the cellulose filter on the reduction of the cryoprotectant during vitrification of mouse embryos. Twenty female mice, four week old,  were used (C57BL / 6 X BalbC) and superovulated by intraperitoneal injection of 10 IU eCG (Equine Chorionic Gonadotropin - Novo-rmon-SYntex®) followed 48h with 10 IU hCG (Chorionic Gonadotrophin human - Vetecor-HertapeCalier®). The protocol suggested by Vitri-Ingá® was used for vitrification and heating.  During the last step of the protocol, the excess of the cryoprotectant medium was aspirated with glass Pasteur pipette (Group A), with filter paper strips (Group B) and cellulose strips (Group C), and immediately mv immersed in liquid nitrogen. Statistical analysis was  performed using the chi² calculation, evaluating survival rates, cleavage, blastocyst formation and hatching. There was no difference between groups A and B on survival rates, cleavage, blastocyst and hatching. However, there were differences (p<0.05) in  blastocyst rates (80.8%, 60%) and hatching (55.3%, 33.3%)  between group A and group C, respectively. It was concluded that the use of filter paper for the vitrification of embryos in the present study was satisfactory and promising, confirming the importance of the cooling rate for the success of the technique and it has also tried to simplify and improve the implementation of the methodology.

Este estudo teve como objetivo avaliar a eficiência do filtro de celulose e papel filtro na diminuição do volume de crioprotetor na vitrificação de embriões murinos. Foram utilizadas 20 fêmeas de camundongos F1 (C57Bl/6 X BALBc), com 04 semanas de idade, e superovuladas por administração intraperitoneal de 10 IU eCG (Gonadotrofina Coriônica equina Novormon-SYntex®) e após 48h com 10 IU hCG (Gonadotrofina Coriônica humana Vetecor-HertapeCalier®). A vitrificação e aquecimento de todos os grupos seguiu o protocolo estabelecido pelo método Vitri-Ingá®. Na última etapa da técnica, o excesso de meio crioprotetor foi aspirado com pipeta pasteur de vidro estirada (Grupo A), com tiras de papel filtro (Grupo B) e com tiras de celulose (Grupo C), sendo imediatamente imersos em nitrogênio líquido. A análise estatística foi realizada através do cálculo chi², avaliando as taxas de sobrevivência, clivagem, formação de blastocistos e eclosão. Percebeu-se que não houve diferença entre os grupos A e B quanto as taxas de sobrevivência, clivagem, blastocistos e eclosão. Houve diferença (p<0,05) nas taxas de blastocisto (80,8%, 60%) e eclosão (55,3%, 33,3%) entre o grupo A e o grupo C, respectivamente. Ao fim, concluiu-se que a utilização do papel filtro na vitrificação de embriões neste estudo foi satisfatória e promissora, confirmando a importância da velocidade de resfriamento para o sucesso da técnica e ainda buscou simplificar e melhorar a execução da metodologia.

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