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Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

Animals↗

Fertilizability of in vitro matured oocytes from golden hamsters.

The fertilizability of hamster oocytes matured in vitro was examined along with two factors potentially affecting nuclear maturation in culture. The four amino acids (isoleucine, methionine, phenylalanine, and glutamine) necessary for nuclear maturation of cumulus-free oocytes (Gwatkin and Haidri, '74) were not required if oocytes recovered on the morning of proestrus (day 4) were cultured with intact cumuli. Although follicular oocytes recovered on day 3 of the estrous cycle (late diestrus) had somewhat lower frequencies of maturation in vitro compared to those recovered on day 4 (76 vs. 95%, respectively), they still had a substantial frequency of spontaneous maturation. Follicular oocytes recovered on day 3 and matured in vitro were fertilized at frequencies equivalent to oviducal oocytes (80 vs. 82%, respectively) when incubation of oocytes with precapacitated sperm was continued for 6 h. Penetration of follicular oocytes was lower (37.4%) after only 4 h of sperm/egg incubation, indicating a delay in sperm penetration with follicular oocytes matured in vitro. Incubation for 4 h is sufficient time for penetration of 80% or more of oviducal oocytes. While 98% of penetrated oviducal oocytes were fertilized normally, only 2% of penetrated follicular oocytes were normal. The majority (85%) of follicular oocytes, unlike oviducal oocytes, were unable to cause decondensation of sperm nuclei after 6 h of sperm/egg incubation. Use of a highly defined system for in vitro fertilization of hamster gametes has provided rigorous proof that isolated cumulus-oocyte complexes do not undergo complete maturation in vitro.

Animals↗

[The influence of follicular factors on the in vitro maturation of bovine oocytes].

Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.

Animals↗

A morphological and functional study of the effect of slow freezing followed by complete in-vitro maturation of primary mouse ovarian follicles.

Mechanically isolated intact early preantral follicles (100-130 microns diameter) from 14 day old mice were cryopreserved by a slow freezing protocol with dimethyl sulphoxide and then matured in vitro for 12 days after rapid thawing. Minor freeze damage observed after 1 day of in-vitro culture included ablation of the theca cell layer and granulosa cell dehydration, resulting in disruption of intercellular contacts with the oocyte and between granulosa cells. Of the follicles, 24% were irreversibly damaged and had a collapsed oocyte. The remaining majority of the follicles had an intact oocyte as evaluated by ultrastructural analysis. Follicles with an intact oocyte were cultured in vitro and, after an initial retarded development, the final number of fully grown oocytes ovulated in vitro was not different from that of unfrozen controls. Cryopreserved early preantral follicles matured in vitro responded to stimulus with human chorionic gonadotrophin in a similar way to controls, with mucification of the oocyte-cumulus complex, germinal vesicle breakdown and extrusion of the first polar body of the oocyte. These cryopreserved, in-vitro matured oocytes had the potential to fertilize and develop to hatched blastocysts.

Animals↗

Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope.

OBJECTIVE: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. DESIGN: Morphological study. SETTING: Academic IVF clinic. PATIENT(S): Women undergoing oocyte retrieval for ICSI treatment. INTERVENTION(S): Oocytes were examined after in vitro maturation. MAIN OUTCOME MEASURE(S): The study examined meiotic spindle morphologic features and chromosome alignments. RESULT(S): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. CONCLUSION(S): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination.

Birefringence↗

Isolation, in vitro maturation, and fertilization of germinal vesicle oocytes obtained from the intact murine ovary.

The purpose of this investigation was to attempt to develop a process, utilizing a murine model, which would allow more efficient harvesting from the intact ovary and maturation in vitro of germinal vesicle (GV) oocytes. The recovery process yielded 25.5 +/- 4.5 (mean +/- SE) cumulus-free GV oocytes per animal. Treatment groups included culture medium (CM) supplemented with either estradiol (E2), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), or prolactin (PRL). Among the hormone-free controls 83.2 +/- 1.6% of oocytes underwent GV breakdown, whereas 25.3 +/- 2.6% developed to the first polar body stage (PB-1) following 18 hr of incubation (n = 29 trials). Oocytes progressing to the PB-1 stage were inseminated in vitro. In vitro fertilization (IVF) of pooled in vitro matured (IVM) PB-1 oocytes (judged by two-cell formation) was 19.9%, which was significantly lower than in the group of in vivo matured oocytes (74.4%). E2 significantly increased the percentage of GV breakdown (control, 76.8 +/- 2.5%; E2 at 10 ng/ml, 92.9 +/- 2.5%, P less than 0.001; E2 at 100 ng/ml, 93.7 +/- 2.1%, P less than 0.001; and E2 at 1 micrograms/ml, 86.7 +/- 3.3%, P less than 0.05) but not PB-1 formation. Neither FSH nor hCG significantly increased GV breakdown or PB-1 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation.

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.

Animals↗

The outcome of ICSI of immature MI oocytes and rescued in vitro matured MII oocytes.

BACKGROUND: The use of immature oocytes is limited to cases where these are the only available oocytes, and they are usually only microinjected with sperm after having undergone maturation in vitro. This study compares the outcome of injection of sperm into metaphase I oocytes immediately after their denudation (MI) performed 2 h after their retrieval, with the outcome of injection of sperm into rescued in vitro matured metaphase II (IVM MII) oocytes after their short incubation in routine laboratory conditions. METHODS: ICSI was performed on MI oocytes, rescued IVM MII oocytes and on MI oocytes that were incubated but failed to extrude their first polar body (arrested IVM MI). Fertilization and cleavage rates were compared with those achieved in mature metaphase II oocytes (MII). RESULTS: ICSI of MI oocytes showed impaired performance compared with ICSI of rescued IVM MII oocytes and MII oocytes, in terms of oocyte degeneration rate (11 versus 6 versus 4%; P < 0.0001), fertilization rate (28 versus 44 versus 68%; P < 0.0001) and multipronucleated fertilization (10 versus 4 versus 4%; P < 0.01). The cleavage rate was lower in rescued IVM MII oocytes compared with MII oocytes (86 versus 95%; P < 0.01). Arrested IVM MI oocytes showed similar results to those of MI oocytes but had a lower cleavage rate (72 versus 96%; P < 0.01). CONCLUSIONS: The injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes.

Adult↗

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries.

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.

Adult↗

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum.

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.

Animals↗

In vitro maturation and fertilization of goat oocytes.

Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17beta. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20 degrees C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%.

Journal Article↗

Serial pronuclear transfer increases the developmental potential of in vitro-matured oocytes in mouse cloning.

In vitro-matured germinal vesicle oocytes are an interesting source of cytoplast recipients in both animal and human nuclear transfer (NT) experiments. We investigated two technical aspects that might improve the developmental potential of nuclear transfer mouse embryos constructed from in vitro-matured germinal vesicle oocytes. In a first step, the effect of two maturation media on the embryonic development of NT embryos originating from in vitro-matured oocytes was compared. Supplementation of the oocyte maturation medium with serum and gonadotrophins improved the developmental rate of NT embryos constructed from in vitro-matured oocytes, but it was still inferior to that obtained with in vivo-matured metaphase II (MII) oocytes. Second, we investigated the effect of serial pronuclear transfer from NT zygotes originating from both in vitro- and in vivo-matured oocytes to in vivo-fertilized zygotic cytoplasts. Blastocyst quality was evaluated by counting nuclei from trophectoderm and inner cell mass cells using a differential staining. Sequential pronuclear transfer significantly improved the blastocyst formation rate of NT embryos originating from in vitro-matured oocytes up to the rate obtained with in vivo-matured MII oocytes. We conclude that the developmental potential of NT embryos constructed from in vitro-matured oocytes can be optimized by serial pronuclear transfer to in vivo-produced zygotic cytoplasts.

Animals↗

Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles.

OBJECTIVE: To describe pregnancies that resulted from in vitro matured oocytes derived from stimulated IVF cycles before cancellation owing to poor response of gonadotropins. DESIGN: Case report. University hospital. PATIENT(S): Eight patients who underwent in vitro maturation.Immature oocyte retrieval, in vitro maturation of immature oocytes, fertilization, and ET. Luteal support with progesterone and plvyeron was given. MAIN OUTCOME MEASURE(S): Pregnancy and live birth. RESULT(S): Three pregnancies (two live births and another ongoing) were achieved after immature oocyte retrieval, in vitro maturation, fertilization with ICSI, and ET. CONCLUSION(S): Immature oocyte retrieval from poor responders during stimulation, followed by in vitro maturation, may be an alternative before the cycle is canceled.

Adult↗

Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation.

The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h (mean +/- S.D.), before being subjected to xenogenous gamete intrafallopian transfer (XGIFT). An average of 13 +/- 0.8 oocytes and 40x10(3) spermatozoa per oocyte were transferred into 20 oviducts of ewes. Fourteen percent of transferred oocytes (36/259) were recovered between 2 and 7 days post-XGIFT and 36% of those recovered displayed embryonic development ranging from the 2-cell to the blastocyst stage. Fertilization following XGIFT was also demonstrated by the detection of zinc finger protein Y (ZFY) loci. Ligation of the uterotubal junction (UTJ), ovarian structures, or the duration of oviductal incubation did not significantly affect the frequency of embryonic development or recovery of oocytes/embryos after XGIFT. In conclusion, equine embryos can be produced in a smaller non-equine species that is easier for handling.

Animals↗

Embryo cloning in cattle: the use of in vitro matured oocytes.

In vitro matured (IVM) bovine oocytes were examined to determine their potential viability in embryo cloning. Activation competence, as monitored by pronuclear formation, increased with oocyte age. Oocytes readily formed a pronucleus when challenged with an electrical pulse 30 h after the onset of maturation. Developmental competence of IVM oocytes tended to increase with oocyte age (P = 0.079). Selection of IVM oocytes on the basis of the presence of a polar body 24 h after the onset of maturation and the size of the follicle from which the oocyte was derived improved development of nuclear transfer embryos (polar body positive 25% versus polar body negative 10%, P < 0.05; large follicle oocytes 31% versus small follicle oocytes 14%, P < 0.05). When selected, IVM oocytes were compared with in vivo matured oocytes recovered from superovulated cows and heifers; no difference was detected for the frequency of embryos produced, pregnancies confirmed between days 50 and 60 of gestation, or the number of calves born. We conclude that selected IVM oocytes are equivalent to in vivo matured oocytes when used for bovine embryo cloning.

Animals↗

Progesterone-induced in vitro maturation in oocytes of Notophthalmus viridescens (Amphibia Urodela) and some observations on cytological aspects of maturation.

Maturation in vitro of oocytes of the newt, Notophthalmus viridescens, is inducible with progesterone after in vivo treatment of females with gonadotropin; few oocytes mature in vitro in the absence of such gonadotropin treatment. Chromosomes of most large oocytes of animals not receiving gonadotropin are still in the lampbrush condition; chromosomes from gonadotropin-treated animals are shorter and the lateral loops are less profuse and somewhat retracted. The chromosome condition, then, can be correlated with susceptibility to progesterone induction of maturation in vitro. As maturation progresses, the germinal vesicle moves toward the surface and decreases in size, with an apparent loss of nuclear material from the centripetal end. Although lateral loops of most chromosomes disappear during the changes in the germinal vesicle, profuse loops develop during this period at the sphere loci, which were previously devoid of loops.

Animals↗

In vitro maturation of oocytes.

In vitro maturation of oocytes is a safe and effective treatment offered in some fertility centers for assisted reproduction, where immature oocytes are retrieved from unstimulated ovaries. Therefore, the procedure avoids ovarian stimulation with expensive gonadotropins, side effects of the medications, and risks such as ovarian hyperstimulation syndrome. Added advantages are reduced frequency of monitoring scans and shorter treatment regimen compared with in vitro fertilization. The candidates initially considered were women with polycystic ovaries having multiple antral follicles, but the indications are widening to include women with primarily poor quality embryos in repeated cycles and poor responders to stimulation. The two new applications for in vitro maturation we are now successfully implementing at McGill Reproductive Center are for oocyte donors and for fertility preservation, especially in women with cancer who are undergoing gonadotoxic therapy. In young women without partners needing this treatment for fertility preservation, it is combined with vitrification of the oocytes. We have achieved a 38% clinical pregnancy rate per cycle in women having IVM for infertility treatment up to the age of 35 years, and 50% clinical pregnancy rate per cycle in recipients of IVM egg donation.

Female↗

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation.

The present study was conducted to determine if the cryopreservation of immature human oocytes has a deleterious effect on the meiotic spindle following maturation in vitro. Oocytes were obtained in excess from in-vitro fertilization patients and divided into four groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immature oocytes cryopreserved before or after maturation in vitro respectively. Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controls and included oocytes matured in vitro and in vivo respectively. The meiotic spindle was identified after incubation in anti-tubulin monoclonal antibody (1 h, 37 degrees C) and fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomes were counterstained with 4',6'-diamidino-2-phenylindole. Following cryopreservation, group 1 oocytes demonstrated a 63% survival rate and 68% maturation rate in vitro. In all, 58% of the oocytes in group 2 survived the thaw. The number of oocytes with normal spindles in group 1 (81.0%) was not significantly different from control groups 3 (83.8%) and 4 (88.9%), while the number of group 2 oocytes with normal structures (43.5%) was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002), and 4 (P = 0.025). These results suggest that cryopreservation of the prophase I human oocyte does not significantly increase abnormalities in the resulting meiotic spindle.

Cell Survival↗