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PubMed2006

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes.

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.

Animals↗

PubMed2001

Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes.

The lipid content and fatty acid composition of fresh immature and in vitro matured bovine oocytes cultured in media with or without serum, and also those of frozen-thawed immature oocytes were analysed. All oocytes were ranked (A or B) on the basis of their cytoplasmic quality. Fatty acid composition (mol %; w/w) in the total lipid fraction was analysed by gas chromatography. Triglyceride, total cholesterol, phospholipid (phosphocholine-containing phospholipid) and non-esterified fatty acid contents of immature and in vitro matured oocytes were determined using lipid analysis kits. Phosphocholine-containing phospholipid and non-esterified fatty acid contents were determined in frozen-thawed immature bovine oocytes. Palmitic acid was the most abundant fatty acid in immature oocytes (A: 35%, B: 36%), and in in vitro matured oocytes cultured in the medium containing serum (A: 36%, B: 35%) or polyvinyl alcohol (A: 33%, B: 36%). Oleic acid was the second most abundant fatty acid in all A ranked oocytes, whereas stearic acid was the second most abundant fatty acid in all B ranked oocytes. There were significant differences (P < 0.05) in linoleic and arachidonic acid fractions between A and B ranked immature oocytes. In vitro matured oocytes had significantly (P < 0.05) lower proportions of linoleic and arachidonic acids, and significantly (P < 0.01) lower contents of triglyceride and total cholesterol compared with those of immature oocytes. The fatty acid composition of in vitro matured oocytes cultured in medium containing fetal calf serum or polyvinyl alcohol was similar, but significant differences (P < 0.01) in triglyceride and the total cholesterol content were observed. There was a significant decrease (P < 0.05) in the arachidonic acid proportion in frozen-thawed immature oocytes compared with that in fresh immature oocytes. In addition, significant (P < 0.05) decreases in both phospholipid (15.8--10.6 pmol) and non-esterified fatty acid (11.0--4.1 pmol) were found in frozen--thawed immature oocytes. The results indicate that lipids are available for use as an energy source for maturation and that serum lipids are incorporated into the oocyte cytoplasm during in vitro maturation. The changes in the lipid content (mainly phospholipid) and fatty acid composition were also observed in frozen--thawed immature oocytes. The study indicates that the alteration of fatty acid composition in bovine oocytes might improve maturation and cryopreservation.

Animals↗

PubMed2006

In vitro maturation of human oocytes for assisted reproduction.

OBJECTIVE: To describe and evaluate the current practice of in vitro maturation of oocytes for assisted reproduction. DESIGN: Review of the available and relevant literature regarding in vitro maturation of oocytes. CONCLUSION(S): In vitro maturation of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies. This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of ovarian hyperstimulation syndrome, and simplicity of protocol. In vitro maturation of oocytes for assisted reproduction in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing in vitro maturation into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

Cell Culture Techniques↗

PubMed1999

In vitro maturation of baboon oocytes retrieved at the time of cesarean section.

OBJECTIVE: To investigate the feasibility of oocyte retrieval at the time of cesarean delivery and the potential of such oocytes to undergo nuclear maturation in vitro using a baboon model and an established culture system. DESIGN: Randomized, controlled animal study. SETTING: Research foundation and university research laboratory. ANIMAL(S): Mature pregnant baboons. INTERVENTION(S): In vitro culture of aspirated oocytes with or without epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Oocyte yield, germinal vesicle breakdown, polar body extrusion. RESULT(S): A total of 246 oocytes were retrieved (mean, 35; range, 14-67). Eighty-seven oocytes (35%) underwent germinal vesicle breakdown and 72 oocytes (29%) extruded a polar body. A chi2 analysis revealed no significant effect of EGF on outcome parameters. No effect of gestational age or maternal age on oocyte yield or development was observed. CONCLUSION(S): A sizeable proportion of oocytes obtained from puerperal primates exhibited the capacity to undergo nuclear maturation in vitro.

Aging↗

PubMed1997

Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes.

In-vitro maturation of human oocytes is an important technique in assisted reproduction due to its potential for reducing the use of fertility drugs. We offered this technique as an alternative to cancelling the cycle to a patient who was at risk of ovarian hyperstimulation syndrome (OHSS) after treatment with gonadotrophin-releasing hormone analogue (GnRHa) and human menopausal gonadotrophin (HMG). The patient had 40 visible antral follicles with a maximum diameter of 13 mm and an oestradiol concentration of 14,000 pmol/l on cycle day 12. Immature oocytes were aspirated transvaginally under ultrasound guidance. Ten cumulus-enclosed oocytes were harvested and nine of them completed nuclear maturation to metaphase II after 48 h in culture. By 18 h after an intracytoplasmic sperm injection (ICSI) procedure, seven of these metaphase II stage oocytes displayed two distinct pronuclei and two polar bodies. All fertilized oocytes but one underwent cleaveage; four of these were transferred 2 days later. Endometrial priming was initiated with 8 mg oestradiol valerate daily from the day of oocyte retrieval and 50 mg progesterone was injected i.m. daily starting 2 days after that. A single intrauterine sac was seen containing one fetus with positive fetal heart beat on ultrasound at 7 weeks of gestation. Unfortunately, the pregnancy ended at 24 weeks shortly after premature rupture of membranes; a live healthy-looking girl was delivered who died 18 days later.

Adult↗

PubMed2005

Metabolic markers of developmental competence for in vitro-matured mouse oocytes.

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus-oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 microl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0-16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8-12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8-16 h of maturation, than by oocytes that did not fertilize (8-12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12-16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

Animals↗

PubMed2000

Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: short communication.

Although strategies for in-vitro maturation of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes, full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.

Animals↗

PubMed1997

Timing of in vivo maturation of equine preovulatory oocytes and competence for in vitro maturation of immature oocytes collected simultaneously.

The objects of this study were to monitor the development of the cumulus complex and nuclear maturation in oocytes recovered from preovulatory follicles following treatment to induce ovulation and to investigate the in vitro maturation competence of oocytes recovered from smaller nonpreovulatory follicles of varying size. All follicles > or =5 mm in pony mares were individually punctured at 0, 6, 12, 24 and 35 h after an injection of LH to induce ovulation. The recovery rates of oocytes were 64% from 55 preovulatory follicles, 22% from 32 subordinate follicles and 52% from 227 small follicles. Cumulus expansion of the preovulatory oocytes occurred at 12 h post LH treatment while the metaphase I and II components of nuclear maturation were not completed until 24 and 35 h post LH respectively. For nonpreovulatory follicles, the frequency of atresia and oocyte competence for in vitro nuclear maturation both increased with increasing follicular size.

Animals↗

PubMed1999

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.

Cell Differentiation↗

PubMed2006

Developmental capacity of Antarctic minke whale ( Balaenoptera bonaerensis ) vitrified oocytes following in vitro maturation, and parthenogenetic activation or intracytoplasmic sperm injection.

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.

Animals↗

PubMed1998

Effect of low temperatures on in-vitro matured bovine oocytes.

This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C or 0 degree C for 5, 10, or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P < 0.05) for oocytes maintained at 39 degrees C (73.2%) than for oocytes cooled at 20 degrees C (58.6%), 10 degrees C (47.3%), or 0 degree C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39 degrees C, 20 degrees C, 10 degrees C and 0 degree C, respectively (P < 0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10 degrees C (0.0%) or 0 degree C (0.9%), followed by 20 degrees C (7.1%) and 39 degrees C (16.5%; P < 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting > or = 2 pronuclei), there was no difference (P > 0.05) between oocytes cooled at 0 degree C (59.7%) or 10 degrees C (67.9%). However, penetration rates in these 2 groups were lower (P < 0.05) when compared to zona-free oocytes cooled at 20 degrees C (83.1%) or those maintained at 39 degrees C (83.1%). Zona-free oocytes had higher penetration rates (P < 0.05) when cooled at 0 degree C (59.7%) or 10 degrees C (67.9%) than zona-intact oocytes cooled at 0 degree C (37.3%) or 10 degrees C (47.2%). However, there was no difference in the penetration rate when zona-free and zona-intact oocytes were cooled at 20 degrees C or maintained at 39 degrees C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida.

Animals↗

PubMed1995

In vitro maturation and fertilization of prepubertal goat oocytes.

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.

Journal Article↗

PubMed1997

Nuclear transfer in sheep embryos: the effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes.

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation if IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 microM nocodazole for 14-17 hr. Three types of transfers were compared directly: "S-->S," early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; "M-->MII," nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S-->MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M-->MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S-->S and M-->MII, and between S-->S and control, though developmental rate was also increased for S-->S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M-->MII or S-->S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M-->MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos.

Animals↗

PubMed2004

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium.

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus-oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7% +/- 5.0%) was significantly (p < 0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p < 0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2% +/- 5.4% vs 57.1% +/- 14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p < 0.05) higher the rate of blastocyst formation (20.0 +/- 5.2%) than that in the control medium (6.2% +/- 3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.

Animals↗

PubMed1992

Zona pellucida surface of immature and in vitro matured mouse oocytes: analysis by scanning electron microscopy.

PURPOSE: The aim of this work was to determine the morphology of the zona pellucida surface of immature and in vitro matured mouse oocytes by scanning electron microscopy. For this purpose two groups of immature oocytes (germinal vesicle group and metaphase I group) were studied either before or after in vitro maturation. RESULTS: Before in vitro maturation, the germinal vesicle immature group showed mainly an unstructured zona pellucida surface with smooth cumulus cells. The metaphase I immature group showed a more structured zona pellucida with smooth or blebbing cumulus cells. After in vitro maturation, development of the zona pellucida toward a mature surface, related to the initial degree of oocyte maturity, was observed in both groups. CONCLUSIONS: These observations show a correlation between the morphology of the zona pellucida surface and the degree of oocyte maturity; the in vitro maturation process can give rise to a proper development of this endowment when immature oocytes are used.

Animals↗

PubMed2004

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity.

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.

Animals↗

PubMed1997

Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare?

In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes to analyse the stages of nuclear maturation. The transfer technique did not impair significantly the final maturation of the recipient preovulatory follicle. The indigenous oocytes within the recipient follicles were recognisable by their larger expanded cumulus of yellow colouration due to high hyaluronic acid content; 7/12 of these oocytes were mature (metaphase II). Around half (42/86; 49%) of the oocytes transferred to preovulatory follicles were recovered subsequently. Most of them showed cumulus expansion (41/42, 6 of which were rich in hyaluronic acid), 13 (32%) were mature, 15 (36%) were immature and 13 (32%) were degenerate. When the indigenous oocyte of the recipient mare was mature, 38% of the transferred oocytes were mature, this rate being no different from the in vitro maturation rate of 46%. This study showed that in vivo maturation of immature oocytes by transfer into a preovulatory follicle in a recipient mare is possible. The maturation rate is not different from the in vitro maturation rate. The technique allows the generation of mature oocytes that have an expanded cumulus rich in hyaluronic acid, similar to the situation in preovulatory oocytes. This result has not been obtained in vitro previously.

Animals↗

PubMed1998

Effects of ovarian source, patient age, and menstrual cycle phase on in vitro maturation of immature human oocytes.

OBJECTIVE: To determine the efficiency of in vitro maturation, expressed by nuclear maturation, of oocytes aspirated during gynecologic surgeries or collected from excised ovaries. To assess the effect of patient age and cycle phase at collection on the oocyte's ability to mature in vitro. To examine the time course of oocyte maturation in vitro. DESIGN: Nuclear maturation based on patient criteria compared. SETTING: University-based IVF program and research center. PATIENT(S): Consented patients undergoing gynecologic surgeries or patients undergoing oophorectomy. INTERVENTION(S): Oocytes were maintained in culture for 48 hours and evaluated for maturation. MAIN OUTCOME MEASURE(S): Nuclear maturation evaluated as germinal vesicle breakdown (GVBD) or progression to the metaphase II (MII) stage. RESULT(S): A significantly higher percentage of oocytes collected during the follicular phase of the menstrual cycle underwent GVBD than did oocytes collected during the luteal phase (60% versus 48%, respectively). The percentage of oocytes reaching the MII stage, from these two groups, was not different. No statistically significant differences in maturation were observed in oocytes from different ovarian sources or from patients >40 or <40 years of age. CONCLUSION(S): These data suggest that oocytes collected during the follicular phase are more likely to undergo GVBD than oocytes collected during the luteal phase. In this study, ovarian source, age, or cycle phase did not influence the final meiotic maturation of oocytes to metaphase II.

Adult↗