RESUMO
BACKGROUND: The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. METHODS AND RESULTS: We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. CONCLUSIONS: We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.
Assuntos
Citoplasma/fisiologia , Desenvolvimento Embrionário e Fetal , Fertilização/fisiologia , Membrana Nuclear/fisiologia , Poro Nuclear/fisiologia , Anticorpos Monoclonais , DNA/metabolismo , Feminino , Imunofluorescência , Humanos , Microscopia Eletrônica , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oócitos/fisiologia , Oócitos/ultraestruturaRESUMO
PURPOSE: To analyze the distribution of a tubulins and acetylated alpha tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. RESULTS: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed > or = 3 pronuclei (PN) and karyomeres after IVF-ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of > or =1 PN within the cytoplasm were the main events present in most oocytes after IVF-ICSI. CONCLUSIONS: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF-ICSI showed similar etiologies in this special group of zygotes.
Assuntos
Citoesqueleto/ultraestrutura , Zigoto/patologia , Acetilação , Adulto , Cromatina/ultraestrutura , Criopreservação , Citoesqueleto/química , Transferência Embrionária , Feminino , Fertilização in vitro , Humanos , Recém-Nascido , Masculino , Meiose , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Síndrome de Hiperestimulação Ovariana/etiologia , Indução da Ovulação/efeitos adversos , Gravidez , Processamento de Proteína Pós-Traducional , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/química , Cauda do Espermatozoide/ultraestrutura , Espermatozoides , Tubulina (Proteína)/análise , Tubulina (Proteína)/química , Zigoto/químicaRESUMO
OBJECTIVE: To establish the predictive value of serum inhibin B levels as an indicator of the presence of testicular spermatozoa in nonobstructive azoospermia, compared with the traditional serum FSH marker. DESIGN: Prospective study. SETTING: Private high-complexity reproductive center with university affiliation. PATIENT(S): Seventy-eight patients with nonobstructive azoospermia, 15 patients with obstructive azoospermia, and 10 fertile volunteers. INTERVENTION(S): Blood samples, testicular sperm extraction, percutaneous epididymal sperm aspiration, and semen collection. MAIN OUTCOME MEASURE(S): Serum levels of inhibin B and FSH and presence of spermatozoa on TESE, PESA, or regular semen analysis. RESULT(S): Patients with nonobstructive azoospermia has significantly higher levels of serum FSH and significantly lower levels of inhibin B. Mean inhibin B serum levels were significantly higher in patients with nonobstructive azoospermia who had spermatozoa on TESE than in those in whom no spermatozoa were found (89.31 +/- 73.24 pg/mL vs. 19.23 +/- 22.34 pg/mL), but mean FSH serum levels did not have similar predictive power (21.37 +/- 12.92 IU/mL vs. 19.27 +/- 10.28 IU/mL). The cut-off level of inhibin B separating both groups, as determined by the receiver-operating characteristic curves, was >53 pg/mL. CONCLUSION(S): Serum inhibin B level seems to be more accurate than serum FSH level in prediction of the presence of testicular spermatozoa in patients with nonobstructive azoospermia.
Assuntos
Inibinas/sangue , Oligospermia/sangue , Testículo/fisiologia , Biomarcadores/sangue , Biópsia , Criopreservação , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Inibinas/fisiologia , Cariotipagem , Masculino , Oligospermia/diagnóstico , Valor Preditivo dos Testes , Gravidez , Estudos Prospectivos , Curva ROC , Preservação do Sêmen , Espermatozoides/fisiologia , Estatísticas não Paramétricas , Testículo/cirurgiaAssuntos
Transferência Embrionária/métodos , Infertilidade Feminina/terapia , Adulto , Gonadotropina Coriônica Humana Subunidade beta/administração & dosagem , Gonadotropina Coriônica Humana Subunidade beta/uso terapêutico , Feminino , Humanos , Indução da Ovulação/métodos , Gravidez , Injeções de Esperma IntracitoplásmicasRESUMO
In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.
Assuntos
Citoesqueleto/ultraestrutura , Fertilização in vitro , Oócitos/ultraestrutura , Cromatina/ultraestrutura , Cromossomos/fisiologia , Citoesqueleto/metabolismo , DNA/ultraestrutura , Feminino , Humanos , Masculino , Metáfase , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitose , Oócitos/metabolismo , Injeções de Esperma Intracitoplásmicas , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Falha de Tratamento , Tubulina (Proteína)/metabolismoRESUMO
AIM: Dysplasia of the fibrous sheath (DFS) is an anomaly found in asthenozoospermic patients with extremely low or absent motility. In order to determine the efficacy of ICSI in these patients, a retrospective analysis of ICSI results in DFS patients has been done. METHODS: Ten ICSI attempts were performed in 6 patients with diagnosis of Dysplasia of the Fibrous Sheath studied by transmission and scanning electron microscopy. RESULTS: In the cases studied, sperm concentration was (29.62 +/- 18.05) x 10(6)/mL, total motility was 1.14 +/- 1.31%. Progressive motility was 0% except for one case with 0.1% . One hundred and three preovulatory oocytes were obtained and 94 metaphase II oocytes were injected. Sixty-nine of them showed two pronuclei (fertilization rate: 73.4%). Forty-nine embryos were obtained and 34 were transferred (mean: 3.4 embryos per transfer). Five pregnancies were diagnosed by beta-hCG plasma level determinations that resulted to be one preclinical abortion, one clinical abortion and three deliveries. Another pregnancy (ongoing) was achieved from a cryopreserved embryo transfer. CONCLUSION: These results showed that ICSI provides a suitable solution for patients suffering from irreversible sperm defects such as DFS. Nevertheless, it is mandatory to inform couples of possible transmission risks to offspring, which are unknown at present. Only when the etiology of this problem is disclosed, it will be possible to assess the real genetic risk.
Assuntos
Transtornos da Motilidade Ciliar , Gravidez/estatística & dados numéricos , Injeções de Esperma Intracitoplásmicas , Espermatozoides , Adulto , Feminino , Humanos , Masculino , Microscopia Eletrônica , Estudos RetrospectivosRESUMO
OBJECTIVE: To report the birth of healthy twin males after the use of testicular spermatozoa from a nonmosaic patient with Klinefelter's syndrome. DESIGN: Case report. SETTING: Private reproduction center with university affiliation. PATIENT(S): A couple undergoing intracytoplasmic sperm injection (ICSI) combined with testicular sperm extraction because of the husband's secretory azoospermia and a nonmosaic 47,XXY peripheral blood karyotype. The wife, a healthy female, presented with a history of oligomenorrhea. INTERVENTION(S): ICSI was performed using testicular spermatozoa; 3 mM pentoxifylline solution was used to induce sperm motility because the spermatozoa recovered were all immotile. MAIN OUTCOME MEASURE(S): Normal fertilization, embryo cleavage, pregnancy outcome, and peripheral blood karyotype of the newborns. RESULT(S): Thirteen metaphase II oocytes were injected. Seven of them fertilized normally and six did not fertilize. Three good-quality embryos (4-cell stage class II) were transferred, and four were cryopreserved at the two-cell and four-cell stages using a slow freezing protocol. Twelve days after ET, a beta-hCG determination was positive. Ultrasonographic examination revealed three intrauterine fetal sacs, but one of them showed a fetal pole without cardiac activity and vanished in subsequent ultrasonographic examinations. The patient delivered twins with normal male peripheral blood karyotypes. CONCLUSION(S): Normal outcome after the use of testicular sperm extraction and ICSI in a nonmosaic patient with Klinefelter's syndrome reaffirms the notion of low transmission risk of this gonosomal aneuploidy.
Assuntos
Fertilização in vitro/métodos , Cariotipagem , Síndrome de Klinefelter/complicações , Microinjeções , Testículo/citologia , Gêmeos , Adulto , Gonadotropina Coriônica Humana Subunidade beta/sangue , Feminino , Humanos , Síndrome de Klinefelter/genética , Masculino , Oligospermia/etiologia , Oligospermia/terapia , Gravidez , Resultado da Gravidez , Espermatozoides/fisiologia , Ultrassonografia Pré-NatalRESUMO
The present report describes a successful intracytoplasmic sperm injection (ICSI) procedure performed with immotile spermatozoa from a young man with a combination of dysplasia of the fibrous sheath and dynein deficiency, a recently described variant of the immotile cilia syndrome. This methodology provides the only suitable solution for these patients in whom all other assisted fertilization technologies have previously failed, and opens the possibilities for treatment of male infertility due to severe, irreversible sperm defects such as the one reported here.
Assuntos
Fertilização in vitro/métodos , Infertilidade Masculina/etiologia , Infertilidade Masculina/terapia , Microinjeções , Doenças Respiratórias/complicações , Espermatozoides/anormalidades , Adulto , Doença Crônica , Dineínas/deficiência , Transferência Embrionária , Feminino , Humanos , Hiperplasia , Hipertrofia , Masculino , Gravidez , Resultado da Gravidez , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/ultraestruturaRESUMO
Comparar los resultados en técnicas de reproducción asistida (ART) empleando dos vias diferentes de administración de análogos de GnRH (diario vs. depósito9