Agonista versus antagonista do GnRH em ciclos de reprodução assistida: DNA fragmentação e apoptose das células da granulosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da resposta inflamatória no soro e líquido folicular em pacientes com anovulação crônica submetidas a hiperestimulação ovariana controlada

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A influência da ativação oocitária artificial com ionóforo de cálcio A23187 em pacientes submetidos a ciclos de injeção intracitoplasmática utilizando diferentes origens de espermatozóides

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

O direito na era das novas tecnologias reprodutivas: modelo jurídico hegemônico e heterogeneidade na parentalidade

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Correlação da maturação e da morfologia do oócito humano com as características do fuso meiótico e da zona pelúcida

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Preservation of wild feline semen by freeze-drying: experimental model

According to the Convention on International Trade in Endangered Species, 36 wild feline species are threatened by extinction or severely endangered, and to save them is the target of several conservation programs. This study aimed to assess the viability of the freeze-drying technique for domestic cat sperm cells, with the ultimate goal of transferring this technology to the wild feline species. The domestic cat is an excellent experimental model for wild felids. It is in this scenario that the freeze-drying process (low-temperature vacuum dehydration) of sperm cells shows its value in preserving male cats' germplasm. Results from membrane and DNA integrity analysis are promising and validates the use of frozen-dried sperm samples in intracytoplasmic sperm injections (ICSIs). Further studies are still necessary to evaluate the ICSI embryo production using domestic cat frozen-dried sperm and the possibility of using such technology with wild felines.

Biotecnologias aplicadas à reprodução de éguas velhas

The acceptance of biotechnology for the most equine breeders association had a significant effect in the horse industry, gaining popularity around the world, because the increasing on the genetic gain, allowing the use of sub fertile mares and stallions with high genetics value on reproduction. The embryos in vitro production of human and cattle has been used with success, however in vitro embryo production is not efficient in the horse, as oocyte transfer (OT) and intracytoplasmatic sperm injection (ICSI). The oocyte transfer has been used especially in subfertile old mares presenting reproductive pathologies as: endometrite, cervical and uterine adhesions, blocked oviduct, perineal laceration and ovulation failures. During oocyte recovery process, the oocytes must be collected from immature follicles that need be matured in vitro or in vivo matured oocytes from pre-ovulatory follicles through the transvaginal aspiration guided by ultrasound. The recovered oocyte is transferred to a previously inseminated recipient mare, through the flank laparotomy. The intracytoplasmatic sperm injection (ICSI) is a procedure of in vitro fertilization that needs only one sperm that is aspirated and injected inside the oocyte. The oocytes used, can be from mature and immature follicles. Fresh, cooled and frozen semen can be used, because the procedure not requires a functional sperm. The use of Piezo drill resulted in a breakthrough the pellucid zone, allowing the vibration per minute provided in the sperm injection pipette, a major result of cleaved oocytes, due to a better sperm injection in the oocyte. The embryo transfer can be straight inside the oviduct, as also transcervical transferred after embryo culture produced in vitro. In conclusion both procedures (OT and ICSI) are effective to be used on equine assisted reproduction, getting results even lower than expected, but satisfactory from animal genetically superior

Injeção Intracitoplasmática de Espermatozoide em Equinos

As técnicas de reprodução assistida utilizadas para os equinos estão em grande desenvolvimento, mesmo com a escassa disponibilidade de ovários de abatedouro para serem feitos estudos e a relação animal/indivíduo entrando, neste caso, a política do bem estar animal e outros fatores que serão discutidos ao longo desta revisão. Podemos afirmar que a técnica de ICSI promove resultados favoráveis à evolução da reprodução assistida, juntamente com outras técnicas, as quais muitas vezes são utilizadas para aprimorar o desenvolvimento e a produção de embriões, não mais necessitando do animal em si e sim de novas técnicas em produção in vitro. Oócito equino tolera o estresse de injeção mecânica de espermatozoides no ooplasma, ou seja, há grande viabilidade da técnica em discussão. O sêmen de baixa qualidade ou com baixo número de espermatozóides pode ser utilizado, seguramente na ICSI, o método pode ser uma ferramenta para avaliar a maturação citoplasmática ou mesmo para substituir outras técnicas já descritas em literatura, mas que não alcançaram a eficiência esperada

Brief Cognitive Behavioral Intervention in Groups in a Brazilian Assisted Reproduction Program

The study's objective was to assess the effect of a cognitive behavioral group intervention on the pregnancy rates of patients submitted to in vitro fertilization (IVF) techniques or to intracytoplasmic sperm injection (ICSI). The study was conducted on 188 patients, 93 who participated in a group of psychological intervention before the IVF and ICSI procedures and 95 patients submitted to IVF and ICSI during the same period of time, who did not participate in the intervention (control group). Clinical pregnancy was the outcome measure. Demographic and clinical variables were compared between groups in order to assess the group's homogeneity. Participants in the psychological intervention obtained a pregnancy rate of 39.8%, significantly higher than the 23.2% rate of nonparticipants (chi(2) = 6.03, p =.01, odds ratio of 22 (CI: 1.16-4.13). The data suggest that group psychological intervention before IVF and ICSI in order to control stress seems to increase the rate of success of these procedures.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida

OBJETIVO: Avaliar o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozoide (ICSI) e comparar os resultados da injeção intracitoplasmática de espermatozoide entre os oócitos em telófase I (TI) e metáfase II (MII), e entre aqueles em metáfase II com e sem fuso celular visível. MÉTODOS: Estudo prospectivo que incluiu 106 pacientes inférteis submetidas à injeção intracitoplasmática de espermatozoide. Foram incluídas pacientes com idade menor ou igual a 38 anos, hormônio folículo estimulante (FSH) basal menor que 10 mIU/mL e índice de massa corpórea (IMC) menor que 30 kg/m². Foram excluídas pacientes com doenças sistêmicas, com qualquer infecção ativa, tabagistas ou que fizeram uso de medicações hormonais e anti-inflamatórias hormonais e não hormonais nos últimos dois meses, previamente à programação para o procedimento de reprodução assistida. Os oócitos com extrusão do primeiro corpúsculo polar foram avaliados pela microscopia de polarização, imediatamente antes da realização da injeção intracitoplasmática de espermatozoide, e caracterizados quanto ao estágio de maturação nuclear (telófase I ou metáfase II). Os oócitos em metáfase II foram avaliados de acordo com a presença ou não do fuso meiótico. Foram analisadas as taxas de fertilização, clivagem e o número de embriões de boa qualidade no segundo dia (D2) de desenvolvimento. Os dados foram analisados comparativamente através do teste exato de Fisher. Em todas as análises foi considerado o nível de significância de 5% (p<0,05). RESULTADOS: O fuso meiótico de 516 oócitos foi visualizado através da microscopia de polarização. Dezessete dos 516 oócitos avaliados estavam em telófase I (3,3%) e 499 (96,7%) em metáfase II. Os oócitos injetados em telófase I apresentaram taxas de fertilização significativamente menores do que os injetados em metáfase II (53 e 78%, respectivamente) e não produziram nenhum embrião de boa qualidade no segundo dia. Comparando-se os oócitos com e sem fuso celular visível, não foi observada diferença significativa nos resultados de injeção intracitoplasmática de espermatozoide. CONCLUSÕES: Oócitos injetados em telófase I apresentaram menores taxas de fertilização quando comparados aos em metáfase II. É possível que a análise do estágio de maturação nuclear oocitária, por meio da microscopia de polarização, possa ser utilizada como fator de predição das taxas de fertilização pós-injeção intracitoplasmática de espermatozoide.

Der Einfluss assistierter Reproduktionstechniken auf die epigenetische Reprogrammierung in der fruehen Embryogenese und Gametogenese im Mausmodell

Seit der Geburt von Louise J. Brown (1978) als erstem künstlich erzeugtem Kind hat sich die Nachfrage nach assistierten Reproduktionstechniken (ART) stark erhöht. Der Anteil der nach In-vitro-Fertilisation (IVF) oder Intrazytoplasmatischer Spermieninjektion (ICSI) geborenen Kinder macht mittlerweile abhängig vom betrachteten Industrieland zwischen 1-4% an der Gesamtgeburtenzahl aus. In zahlreichen Studien korreliert eine erhöhte Prävalenz für seltene Imprinting-Erkrankungen, wie z.B. Beckwith-Wiedemann oder Angelman-Syndrom, mit der Geburt nach assistierten Reproduktionstechniken. Es ist bekannt, dass die medizinischen Interventionen zur Behandlung von Sub- und Infertilität in sehr sensitive Phasen der epigenetischen Reprogrammierung des Embryos und der Keimzellen eingreifen. In der vorliegenden Arbeit wurde untersucht, ob die ovarielle Stimulation einen Einfluss auf die epigenetische Integrität von geprägten Genen in murinen Präimplantationsembryonen hat. Die in diesem Zusammenhang entwickelte digitale Bisulfitpyrosequenzierung gewährleistet die Analyse der DNA-Methylierung auf Einzelallelebene durch eine adäquate Verdünnung der Probe im Vorfeld der PCR. Die ovarielle Induktion führte zu einem erhöhten Rate an Epimutationen des paternalen H19-Allels, sowie des maternalen Snrpn-Allels. Zudem konnte festgestellt werden, dass die Expression von drei potentiellen Reprogrammierungsgenen (Apex1, Polb, Mbd3) in Embryonen aus hormonell stimulierten Muttertieren dereguliert ist. Whole-Mount Immunfluoreszenzfärbungen für APEX1 korrelierten dessen differentielle Genexpression mit dem Proteinlevel. Anzeichen früher apoptotischer Vorgänge äußerten sich in Embryonen aus hormonell induzierten Muttertieren in der hohen Rate an Embryonen, die keines der drei Transkripte exprimierten oder weniger APEX1-positive Blastomeren aufwiesen.In einer weiteren Fragestellung wurde untersucht, ob die Kryokonservierung muriner Spermatozoen den epigenetischen Status geprägter Gene in den Keimzellen beeinflusst. Die Analyse von F1-Zweizellembryonen, die durch IVF mit den jeweiligen Spermatozoen eines Männchens generiert wurden, diente der Aufklärung möglicher paternaler Transmissionen. Insgesamt konnten keine signifikanten Auswirkungen der Kryokonservierung auf den epigenetischen Status in Spermatozoen und F1-Embryonen ermittelt werden.

Anti-Müllerian hormone and inhibin B as predictors of pregnancy after treatment by in vitro fertilization/intracytoplasmic sperm injection

OBJECTIVE: To evaluate anti-Müllerian hormone (AMH) as a marker of reproductive outcome after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Longitudinal study. SETTING: University hospital. PATIENT(S): Two hundred seventy-six consecutive women undergoing IVF/ICSI. INTERVENTION(S): Ovarian stimulation, oocyte retrieval, IVF, ICSI, embryo transfer, AMH, and inhibin B determinations in serum and follicular fluid (FF). MAIN OUTCOME MEASURE(S): The AMH and inhibin B concentrations in 276 matched FF/serum pairs have been determined. Different outcome groups have been compared and set in relation to the oocyte count, morphological parameters, and steroid hormone levels. RESULT(S): The concentrations of AMH and inhibin B in both serum and FF were significantly higher in the group of women who became pregnant in the corresponding treatment cycle than in those who did not conceive. Positive correlations were observed between serum inhibin B concentrations and embryo morphology (r = 0.126, 95% confidence interval 0.026-0.284). Serum and FF AMH or inhibin B correlated positively with the oocyte count and negatively with the pretreatment cycle day 3 FSH level and the total administered gonadotropin dose. CONCLUSION(S): The AMH and inhibin B levels on the day of oocyte retrieval are correlated to reproductive outcome.

Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase

OBJECTIVE: To analyze if oocytes can be obtained in all patients before cancer treatment within 2 weeks by initiating ovarian stimulation during the follicular or luteal phase. DESIGN: Prospective controlled multicenter trial. SETTING: Four university-based centers. PATIENT(S): Forty cancer patients before chemotherapy. INTERVENTION(S): Twenty-eight patients were stimulated with gonadotropins in the follicular phase (group I). In 12 patients (group II), ovarian stimulation was initiated in the luteal phase, and these received GnRH antagonists and recombinant FSH. In 14 patients, 143 oocytes were further processed for fertilization by intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Number of oocytes aspirated after ovarian stimulation, cumulative FSH/hMG dosage, viability and maturity of oocytes, and fertilization rate by ICSI. RESULT(S): Patients in group I (age 27.6 +/- 4.9 yrs) were stimulated on average for 10.6 days, and patients in group II (age 31.2 +/- 5.7 yrs) for 11.4 days. Total amount of FSH was on average 2,255 IU (I) and 2,720 IU (II) per patient. Average and median numbers of aspirated oocytes were, respectively, 13.1 and 11.5 (I) versus 10.0 and 8.5 (II); 83.7% (I) and 80.4% (II) of the oocytes were mature and viable and could be treated by ICSI. Fertilization rate was 61.0% (I) versus 75.6% (II). CONCLUSION(S): This pilot study suggests that oocytes can be obtained before cancer treatment efficiently irrespective of the phase of the menstrual cycle.

Vocabulary and language model adaptation using information retrieval

International audience