Search PubMed⌕ Search

Biomedical subjects

D Nogueira

Publications and source records attributed to D Nogueira.

At least 19 recordsLinked to original sources
PubMed2005

Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Immature oocyte in-vitro maturation: clinical aspects.

The development of immature oocyte collection techniques for in-vitro maturation (IVM), combined with novel culture techniques, opens new possibilities for assisted reproductive technology. Optimization of clinical management of IVM cycles will enhance pregnancy outcome, so that IVM might become an effective alternative assisted reproduction treatment for infertile patients irrespective of the cause of infertility. Parameters such as age and baseline antral follicular count are predictive of outcome and should be used as selection criteria for IVM treatment. Women with polycystic ovary disease and normo-ovulatory patients at risk of developing ovarian hyperstimulation syndrome might benefit from earlier retrieval of oocytes followed by IVM and embryo transfer. HCG priming before oocyte retrieval seems beneficial in terms of oocyte yield and maturational competence, and may increase the harvest of mature oocytes and lead to better endometrial synchronization with the developing embryo. The timing of aspiration may be crucial in IVM and selection criteria for follicle size at aspiration need defining prospectively for infertility type. Finer calibre aspiration needles and low aspiration pressure yield more oocytes. A combination of natural cycle IVF with IVM is a promising, mild and inexpensive assisted reproduction treatment, widely accessible the infertile population.

Animals↗
PubMed2005

Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development.

Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 microM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

Animals↗
PubMed2002

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production.

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.

Animals↗
PubMed2001

Avoidance of multiple pregnancies after ovulation induction by supernumerary preovulatory follicular reduction.

OBJECTIVE: To evaluate the effect of supernumerary preovulatory follicular reduction as an approach to avoid multiple pregnancies in ovulation induction or superovulation cycles. DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENT(S): In 26 cycles, 24 patients underwent ovulation induction or superovulation with either clomiphene citrate or hMG. INTERVENTION(S): Selective follicle aspiration was performed before hCG administration. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate and numbers of multiple pregnancies. RESULT(S): A mean number of 4.5 follicles with a diameter > or =15 mm and a mean number of 4.5 follicles with a diameter < or =14 mm were observed before hCG administration. A mean number of 2.3 follicles with a diameter > or =15 mm and a mean number of 1.8 follicles with a diameter < or =14 mm were aspirated before the hCG administration. Seven singleton pregnancies (26.9% per cycle) ensued from the treatment. CONCLUSION(S): Aspiration of supernumerary follicles after ovulation induction or superovulation seems to be a valid approach to avoid multiple pregnancies without affecting pregnancy rate.

Adult↗
PubMed2001

Improving in vitro maturation of oocytes in the human taking lessons from experiences in animal species.

One to three per cent of infertile women develop severe ovarian hyperstimulation syndrome after superovulation for assisted reproduction treatment (ART). This severe complication can be avoided when oocytes are obtained at an immature stage (germinal vesicle stage) out of small or medium-sized follicles. This hypothesis has been tested in several infertile women, but clinical pregnancies are disappointlingly low. This new approach in ART is still at an experimental phase and this treatment has still to be improved before routine clinical application. Experimental work in animals and humans suggest a beneficial effect in providing a short preliminary pretreatment with follicle-stimulating hormone to select for a developing cohort of follicles. The aspiration of oocyte cumulus complexes is carried out with a short needle applying reduced aspiration pressure. A crucial point is to provide the appropriate culture environment for the immature oocytes. An optimal cumulus-enclosed human oocyte culture system needs to be defined. The composition of the culture medium could be suggested by in vitro work carried out in animal models. As developmental competence is established during the latest phases of oocyte growth and is dependent on the storage of RNA, a prolonged in vitro maturation period (before inducing nuclear maturation) could provide the necessary transcriptional and translational changes. The conditions to achieve this improved cytoplasmic maturation by prolonging the in vitro culture remain to be defined. More objective noninvasive parameters for oocyte maturity are also needed to pursue research in this field.

Animals↗
PubMed2001

Confocal microscopy: principles and applications to the field of reproductive biology.

Confocal microscopy allows analysis of fluorescent labeled thick specimens without physical sectioning. Optical sections are generated by eliminating out-of-focus fluorescence and displayed as digitalized images. It allows 3-dimensional reconstruction (XYZ) and time-analysis (XYT), thus providing unique chance to link morphology with cell function. Since images are obtained by scanning, excess illumination of the specimen and quick decrease of the fluorescent signal are avoided. Resolution obtained with a Laser Scanning Confocal Microscopy (LSCM) is theoretically better than that of a conventional microscope. The preparation of the specimen may be based on standard techniques, such as immunocytochemistry applied to fixed cells, or on staining of living cells, following the use of different fluorescent probes at the same time (colocalization). In our laboratory, we use the LSCM system Fluoview version 2.1 (Olympus) to study reproductive biology of animals and humans. We work on stainings of oocytes and blastocysts (mouse, bovine, human), and human ovarian tissues. We study mitochondrial distribution, cortical granule migration, calcium oscillations and spindle quality to link culture conditions and oocyte quality. Staining of F-actin is used to check transzonal projections (in zona pellucida) or to detect abnormalities following experimental treatment. Blastocyst quality is analyzed in sequential optical sections for microfilament organization and counting of total cell number (staining with phalloidin (actin) and picogreen (DNA). Trophectoderm and inner cell mass distribution (differential staining), apoptotic cells (TUNEL method) and viable cells (live/dead test) are also evaluated. Confocal imaging can be helpful for rapid determination of follicle density (staining with AM Calcein) and follicle morphology (picogreen) in ovarian cortical biopsies. The current review describes the principles of confocal microscopy and illustrates its applications to the field of reproductive biology by a large collection of pictures.

Animals↗
PubMed2000

Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes.

OBJECTIVE: To analyze embryos after in vitro maturation by investigating their nuclear status and cytogenetic constitution. DESIGN: Prospective randomized laboratory study. SETTING: Reproductive medicine unit in an academic hospital. PATIENT(S): Patients with male and tubal factor infertility undergoing fertility treatment. INTERVENTION(S): Denuded immature oocytes (n = 75) were matured in vitro for 24-30 hours, and intracytoplasmic sperm injection was performed 30 hours after oocyte retrieval. Fluorescence in situ hybridization was performed on the produced embryos. MAIN OUTCOME MEASURE(S): Blastomere content of the total embryo. RESULT(S): The in vitro-matured oocytes showed a similar fertilization rate as the in vivo-matured oocytes, but with a higher incidence of noncleavage (21.0%). In addition, 26.7% of these embryos arrested at the first mitotic division. Thirty embryos were processed for fluorescence in situ hybridization; only 6.7% had all mononuclear blastomeres, 30.0% had at least one binuclear blastomere, 43.3% had at least one multinuclear blastomere, and 56.6% contained anuclear cells. The chromosomal constitution was analyzed in 14 embryos, and chromosomal anomalies were found in 11 (78.5%). CONCLUSION(S): Germinal vesicle oocytes retrieved from superovulated patients and cultured in vitro for a short time had the ability to resume meiosis and achieve fertilization. However, arrest of embryo development was common. These embryos showed a high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities.

Adult↗
PubMed1999

Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies.

The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen-thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.

Adult↗
PubMed1998

Ultrastructure of gametes after intracytoplasmic sperm injection.

The ultrastructure of 18 metaphase-II oocytes after intracytoplasmic sperm injection (ICSI) was analysed from 30 min to 8 h following the microinjection. Three control metaphase-II oocytes were not injected. The 21 oocytes were embedded individually and examined in serial pole-to-pole sections. Seventeen oocytes had been matured in vitro from the germinal vesicle stage and showed ultrastructural alterations due to long-term culture. In microinjected oocytes, membrane-bound vacuoles and oolemma inclusions were found at the injection site. The ICSI oocytes showed evidence of plasma-membrane damage and increased oocyte exchange processes with residual and multivesiculated bodies. Gamete membrane fusion was not observed. Acrosome reaction was observed within the ooplasm 15 min after ICSI. Sperm elements were not incorporated into any oocyte vacuole. Onset of oocyte activation was associated with sperm chromatin decondensation. Cortical granules were released at 60 min and two pronuclei were formed at 6 h after ICSI. The male and female pronucleus formation was asynchronous. The findings suggest specific ICSI-related morphological features of early fertilization.

Cytoplasm↗
PubMed1975

Variable-column bubble oxygenator. A new system for bubble oxygenation.

A new bubble oxygenator has been developed. Its main advantage is that the dimensions of the oxygenating chamber can be varied. Thus, the capacity for oxygenation may be adapted to different patients. It was used in 2,700 open-heart operations performed at the Institut do Coracão, Faculdade de Medicina, Universidade de São Paulo, between January, 1971, and December, 1973. A group of 50 patients were subjected to more detailed studies. We found that the average hemolysis was 38.67 mg. per cent with a mean perfusion duration of 69 minutes, 38 seconds. Fibrinogen and platelet concentrations decreased by 14 and 25 per cent, respectively. The pH, PcO2, PO2, and base excess before and after cardiopulmonary bypass ranged within normal levels, and oxygenation (PO2) was always satisfactory.

Adolescent↗