Development of in-vitro-derived bovine embryos cultured in 5% CO2 in air or in 5% O2, 5% CO2 and 90% N2

To evaluate the effects of a three gas mixture of 5% O2, 5% CO2 and 90% N2 (OCN) on preimplantation embryo development, bovine in-vitro fertilization (IVF) oocytes were cultured in a defined medium (mBECM) with various supplements either under 5% CO2 in air or under OCN. When cultured in mBECM alone, embryo development was significantly stimulated in OCN compared to 5% CO2 in air (experiment 1). In the OCN atmosphere, blastocyst formation was further increased after addition of fetal bovine serum (FBS; 10%) or FBS + cumulus granulosa cells (CGC) to mBECM. The ratio of blastocysts to 8-cell embryos, number of hatched blastocysts and embryo diameter were markedly increased, and zona thickness was decreased after FBS addition. However, development up to the morula stage was fully supported by mBECM alone. There was no significant effect of beta-mercaptoethanol (ME; 10 microM) in OCN. In the 5% CO2 atmosphere, embryo development was significantly (P < 0.05) enhanced after addition of FBS + CGC + ME. In experiment 2, in OCN, FBS added at 60 h post-insemination was effective in stimulating blastocyst formation, but changes in medium volume per oocyte from 13.6 to 1.36 microliters had only a marginal effect. In conclusion, OCN gas mixture provides a suitable atmosphere for early embryo growth in vitro and mBECM + FBS in the optimal culture medium under this atmosphere.     

Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development

One of the major morphological anomalies observed in many human pre- embryos is extensive cellular fragmentation. Previously we confirmed that embryo fragmentation seemed to be associated with the activation of programmed cell death (PCD). The purpose of our experiments was to establish a rate for murine embryo fragmentation in vivo after hormonal stimulation in young versus older females and to compare it with the rate of embryo fragmentation during in-vitro fertilization (IVF). While murine maternal age beyond 40 weeks increased the rate of embryo fragmentation following in-vivo fertilization (P = 0.001), oocytes from females of all ages had a uniformly high rate of fragmentation when fertilized in vitro (33%). None of the fragmented murine embryos proceeded further in development. In the mouse, fragmentation occurs exclusively during the first cell cycle. Furthermore, IVF significantly reduced the rate of blastocyst formation (P = 0.0001) and decreased the mean cell number at the blastocyst stage in comparison with embryos produced in vivo (P < 0.0001). The cell death index was significantly affected by both maternal age (P = 0.005) and IVF (P = 0.0001). Identification of specific factors which trigger PCD, especially those associated with IVF, may enable us to lower the rates of fragmentation in preimplantation embryos and thereby increase pregnancy rates after human IVF.      

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection

The aim of this study was to examine the relationship between different preincubation periods of oocytes and the outcome of intracytoplasmic sperm injection (ICSI). We analysed retrospectively 95 ICSI treatment cycles performed to alleviate severe male-factor infertility. Oocyte collection was performed approximately 36 h after human chorionic gonadotrophin administration. The cumulus-corona-oocyte complexes obtained were incubated until the moment of ICSI. Fertilization, embryo development and implantation rates were analysed in four groups, which were divided according to the time lapse between oocyte retrieval and ICSI: group I, < or =3 h (18 cycles); group II, >3-< or =6 h (52 cycles); group III, >6-< or =9 h (14 cycles); and group IV, >9-< or =12 h (11 cycles). Immediately before ICSI the cumulus and corona cells were removed from the oocytes. A total of 723 metaphase II oocytes were injected: 126 from group I, 380 from group II, 126 from group III and 91 from group IV. The fertilization rates obtained were 52.3, 66.8, 65.1 and 69.2% respectively [P < 0.05 (using the chi2 test) between group I and groups II, III and IV]. Cleavage rates were similar in all groups (68.1, 69.7, 79.2 and 79.3% respectively), but the proportion of good quality embryos (< or =20% fragmentation) was significantly lower (P < 0.05) in group I (24.2%) compared with groups II (39.8%) and IV (39.6%). However, no statistically significant differences were observed between the four groups with regard to implantation rates (11.7, 13.2, 10.4 and 20.4% respectively). The results suggest that a preincubation period between oocyte retrieval and ICSI can improve the fertilization rate and embryo quality. This period might be necessary for some oocytes to reach full cytoplasmic maturity, leading to a higher activation rate upon microinjection.      

In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells

Human zygotes resulting from IVF were placed in two differentculture systems to evaluate in-vitro development and to establishpregnancies in patients following embryo replacement. TreatmentA (control) consisted of culturing zygotes in a modified Earle'sBalanced Salt solution while treatment B consisted of culturingzygotes on a monolayer of fetal bovine uterine fibroblasts inthis same culture medium. At the time of embryo replacement,embryos within treatments A and B had 3.7 and 4.3 blastomerespresent, respectively. After 24 h in culture, the cellular fragmentationrate for treatment A embryos was 0.85 which was greater (P <0.05)than the fragmentation rate of 0.44) for embryos within treatmentB. The incidence of implantation for patients whose embryoswere given treatment A was 17.0% which was lower (P <0.05)than 35% for those given treatment B. Implantation rates increasedwith time in culture (43%) for treatment B embryos. Cultureby treatment B of three pronucleate zygotes resulted in 7/9and 4/9 reachIng the blastocyst and expanded blastocyst stages,respectively, whereas only 1/26 three-pronucleate zygotes culturedusing treatment A reached either of these stages.     

First polar body morphology and blastocyst formation rate in ICSI patients

BACKGROUND: It may be beneficial to identify, at a very early stage of development, concepti that will result in viable blastocysts by using a non-invasive technique. METHODS: Homogeneous groups in terms of first polar body (PB) morphology were analysed with regard to fertilization, embryo quality and blastocyst formation. The strategy was to transfer a maximum of two blastocysts with an adequate inner cell mass deriving from oocytes with identical first PBs in order to obtain information about the actual implantation potential. RESULTS: A significant relationship between first PB morphology and embryo quality was found. Fragmentation after 2 days was increased in embryos derived from oocytes with fragmented first PBs (P < 0.05) in comparison with those derived from oocytes with intact PBs. No similar correlation could be demonstrated for fertilization rate. Embryos in the intact first PB group showed an increased rate of blastocyst formation as compared with the fragmented first PB group (P < 0.05). In addition, a significant difference in implantation rate (48.6 versus 22.0%; P < 0.025) and ongoing pregnancy rate (68.4 versus 34.8%; P < 0.05) was observed for the intact versus fragmented groups respectively. CONCLUSION: In conclusion, the current study provides further evidence that preselection at a very early stage may be helpful in identifying a subgroup of preimplantation embryos with a good prognosis to form blastocysts and, consequently, to implant.     

Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts

Culture of in-vivo-developed mouse blastocysts in a simple culture medium based on a balanced salt solution supplemented with carbohydrates for 3 h significantly perturbed embryo metabolism. Maximal perturbation occurred after just 6 h of culture. Similarly, culture of rat blastocysts in a simple culture medium for 3 h also resulted in perturbed metabolism. Cultured mouse and rat blastocysts both had an abnormally elevated rate of glycolysis of approximately 100% after culture (P < 0.05). Rates of pyruvate oxidation by mouse blastocysts were also significantly reduced after culture in a simple medium for 6 h (P < 0.01). Furthermore, the developmental competence of mouse blastocysts after transfer was significantly reduced by just 6 h of culture in a simple medium (P < 0.05). Addition of Eagle's amino acids or vitamins to the culture medium reduced the perturbation of both the glycolytic activity and oxidative capacity of cultured mouse blastocysts and acted in synergy to further the inhibition. Importantly, culture with amino acids and vitamins prevented any loss of viability of mouse blastocysts after culture for 6 h. It can be concluded that the mouse blastocyst is sensitive to its environment and that culture-induced stress results in the loss of normal cellular function, as manifested in this case by an abnormal pattern of glucose utilization and loss of viability.      

Protein kinase C plays an important role in the human zona pellucida- induced acrosome reaction

To investigate the involvement of protein kinases in signal transduction in the human zona pellucida (ZP)-induced acrosome reaction (AR), the effects of protein kinase (PK) activators, dibutyryl cAMP (PKA) and cGMP (PKG), phorbol 12-myristate 13-acetate (PMA, PKC), and the PKC inhibitor, staurosporine were studied. Sperm samples were obtained from normozoospermic men with normal sperm-ZP binding. Oocytes were obtained from other patients with failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were pre- incubated with 2.5-20 microM PMA, 1 mM dibutyryl cAMP or cGMP, 3 mM pentoxifylline or 0.125-2.0 microM staurosporine for 30 min and then incubated with four oocytes for 2 h in human tubal fluid supplemented with bovine serum albumin. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small bore pipette and the state of the AR was determined by fluorescein-labelled Pisum sativum agglutinin. Motility and movement characteristics were assessed by computer-assisted sperm analysis (CASA) after incubation of spermatozoa with PMA for 30 min and 2 h. The dibutyryl cAMP and cGMP analogues had a small positive effect (P < 0.05) but pentoxifylline had no effect on stimulating the ZP-induced AR (P > 0.05). In contrast, PMA stimulated ZP-induced AR in a marked dose-dependent manner. Only the highest concentrations (15-20 microM) of PMA significantly decreased percentage motility (P < 0.001). Doses of 2.5-15 microM of PMA significantly stimulated ZP-induced AR without decreasing motility (P < 0.001). The PKC inhibitor, staurosporine (0.125-0.25 microM) significantly inhibited ZP-induced AR without affecting motility (P < 0.001). Sperm samples from 33 normozoospermic men were used for studies of the ZP-induced AR augmented with 15 microM PMA. One sample did not show a response to PMA stimulation. Among the 14 men with low ZP- induced AR, half had normal responses to PMA and other half had low responses to PMA. In conclusion, activation or inhibition of PKC significantly increases or decreases human ZP-induced AR suggesting that PKC plays a important role in the signal transduction pathway for the physiological AR.      

Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes

This study aims (i) to ascertain whether oxidative-stress-induced disturbances in chromosomal distribution in the metaphase-II spindle of mouse oocytes can be counteracted by supplementing culture medium with antioxidants; and (ii) to determine whether supplemental intake of antioxidants neutralizes the disturbing effects of maternal ageing on segregation of chromosomes during the first meiotic division and distribution of chromosomes in the metaphase-II spindle. (i): Germinal vesicle oocytes from unstimulated 10-12 week old mice were matured in vitro in the presence or absence of diamide and/or dithiothreitol. Metaphase-II oocytes were fixed and stained with 4',6-diamidino-2- phenylindole (DAPI) to detect abnormalities in chromosomal distribution. The percentage of oocytes arrested in metaphase I (12.9% vs 28.4%; P < or = 0.05) or with a telophase-I chromosome configuration (0.0% vs 8.2%; P < or = 0.0005) was decreased in diamide-DTT-treated oocytes when compared to diamide-treated oocytes. (ii): Mice were fed, from the first day of weaning until their death, a diet supplemented or not with an antioxidant mixture of vitamin C and vitamin E. Ovulated oocytes were fixed and stained with DAPI or C-banded for chromosome analysis. The percentage of abnormal (chromosome scattering and nulloploidy) or asynchronous (anaphase I or telophase I) oocytes was 2.7-fold higher in controls than in females fed an antioxidant diet (24.4% vs 8.9%, P < or = 0.05). Furthermore, the percentage of aneuploidy (2.2% vs 0.0%; P < or = 0.01) and diploidy (5.8% vs 1.7%; P < or = 0.05) was significantly higher in controls than in females fed an antioxidant diet. These findings support Tarin's oxidative stress hypothesis of aneuploidy and have clinical implications for preventing both laboratory-induced and maternal-age-associated aneuploidy in human beings.      

Gamete-specific DNA fragmentation in unfertilized human oocytes after intracytoplasmic sperm injection

The objective of the present study was to assess the integrity of maternal and/or paternal chromatin in injected oocytes that remained unfertilized after intracytoplasmic sperm injection (ICSI). The study was performed on 102 oocytes that failed to show pronuclear formation 18-20 h after ICSI. We used chromatin labelling with 4,6-diamidino-2- phenylindole (DAPI) to identify maternal and paternal chromatin, coupled with biotin-mediated end-labelling to assess DNA fragmentation in each gamete. It was shown that 50% of oocytes without pronuclear formation following ICSI contained chromatin with damaged DNA, and that the source of the DNA fragmentation was equally divided between the spermatozoon (25.8%) and the oocyte (24.4%). A significantly greater proportion of condensed spermatozoa in human oocytes had damaged DNA, compared to decondensed spermatozoa (24.7 compared to 5.9%, P=0.002). There was a significant increase in the incidence of DNA fragmentation in oocytes from patients older than 35 years (65+/-1.2%) compared to those from younger patients (36+/-1.0%) (P < 0.05). Further, 17% of unfertilized oocytes contained no paternal chromatin. Thus, DNA fragmentation in both spermatozoa and oocytes is associated with failure of fertilization in ICSI. In some cases of severe male factor infertility, a significant proportion of spermatozoa injected into oocytes may contain fragmented DNA. Injection of oocytes with spermatozoa containing abnormal chromatin will probably result in failure of sperm decondensation and fertilization. In older women, a significant proportion of oocytes injected may contain fragmented DNA. These observations may explain the consistent inability of most clinics to achieve fertilization rates higher than 65% with ICSI.      

Characterization of telomerase activity in the human oocyte and preimplantation embryo

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.     

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.      

Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration

BACKGROUND: Recently, interest in oocyte cryopreservation has steadily increased. Newly developed protocols have dramatically improved survival rates, removing perhaps the major hurdle that has prevented this approach from becoming a fully established form of treatment. However, the clinical efficiency of these protocols has not been exhaustively explored and therefore remains controversial. METHODS: Morphologically normal oocytes displaying the first polar body were frozen-thawed with a slow cooling protocol that utilized 1.5 mol/l propane-1,2-diol (PrOH) and 0.3 mol/l sucrose. RESULTS: A total of 927 oocytes from 146 patients were frozen-thawed, achieving a 74.1% survival rate. Over 76% of microinjected oocytes displayed two pronuclei 16 h post-insemination, while the proportion of embryos at 44-46 h post-insemination was 90.2%. At this time point, the majority (68.3%) of embryos were at the two-cell stage, showing in most cases (78.7%) minimal or moderate fragmentation. Eighteen clinical pregnancies, three of which were twin, were observed, giving rise to rates of 12.3 and 9.7%, calculated per patient and per embryo transfer, respectively.The implantation rate was 5.2%. To date, four children have been born and three pregnancies resulted in spontaneous abortions, while the remaining pregnancies are ongoing. CONCLUSIONS: Our data indicate that although the combination of slow cooling and high sucrose concentration ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency.     

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.      

Developmental fate of ovoid oocytes

BACKGROUND: Irregularities in composition, thickness and/or color of the zona pellucida may impair optimal function and result in reduced outcome. Anomalies of oocyte shape have not been investigated in detail in this respect. METHODS: Therefore, all patients attending our clinic within a period of 1 year were screened for the presence of ovoid gametes and the corresponding developmental potential was evaluated. For all elongated gametes, a roundness index (RI; length divided by width) was calculated in order to quantify shape. RESULTS: RI did not affect fertilizability (P > 0.05). The degree of dysmorphism was found to be related to cleavage pattern. The more ovoid a gamete was, the higher was the risk of the corresponding zygote not cleaving like a tetrahedron (P < 0.01). Abnormal cleavage (a rather flat array of blastomeres) was associated with delayed compaction (P < 0.01) and blastocyst formation (P < 0.001). The quality of blastocysts was not affected at any stage in ovoid concepti (P > 0.05). CONCLUSIONS: Ovoid oocytes with abnormal cleavage pattern show delayed preimplantation development, probably due to a reduced number of cell-to-cell contacts.     

Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids.

BACKGROUND: We modified the loading of pulled straws into a new closed system, called closed pulled straws (CPS) for holding oocytes for vitrification. The morphological survival, dynamics of meiotic spindles, and fertilization in vitro of vitrified oocytes using CPS were compared with conventional straws, open pulled straws (OPS), and grids. METHODS: Surviving oocytes were stained for spindles and chromosomes after 1, 2 and 3 h incubations, and compared with controls. The capacity of fertilization and embryonic cleavage were examined in vitro. RESULTS: The survival rates of the CPS (79%) and straw (77%) groups were significantly higher (P < 0.05) than the OPS (63%) and grid (39%) groups. At a 1h incubation, vitrified oocytes of four groups had significantly fewer normal spindles than controls (P < 0.05). The straw group was inferior to the others in spindle morphology (P < 0.05). After a 3 h incubation, recovery of vitrified oocytes with normal spindles was significantly improved in all groups (P < 0.05). The percentages of fertilization and blastocyst formation of vitrified oocytes after a 1 h incubation was significantly lower than controls (P < 0.05), but they were improved after 2 or 3 h incubations (P < 0.05). CONCLUSIONS: Oocytes vitrified using CPS, OPS or grids could lessen spindle injuries and expedite recuperation. The survival using OPS or grids is lower. Sufficient culture time for recovery of meiotic spindle would be imperative for fertilization events of vitrified oocytes. CPS has the advantages of achieving a high survival and preserving good spindles.     

Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells

In a prospective randomized study, excess embryos from 100 womenundergoing in-vitro fertilization were cultured from the 2-cellto the hatched-blastocyst stage in the presence or absence ofa confluent monolayer of Vero cells. The frequencies of fragmentation,developmental arrest, multi-nucleation and blastocyst formationwere observed for 254 embryos over 7 days in culture. The numberof nucleated cells, and fine structure of trophectoderm andinner cell mass were analysed at the expanded blastocyst stageon day 5.5 post-insemination. The frequency of hatching fromthe zona pellucida was determined between days 6 and 7 post-insemination.With respect to these developmental parameters, the findingsindicate that no overt or statistically significant improvementin early human embryogenesis occurs in the co-culture system.     

Steroid Production by the cumulus: relationship to fertilization in vitro

Insemination media were Collected from 92 follicles of 14 patientsstimulated to progesterone and oestradiol in the inseminationdrops were assayed, corrected for carry–over from follicularfluid and volume and expressed as production per µg ofprotein in the cumulus. significantly higher progesteron productionper unit protein was associated with oocytes which fertilizedin vitro (P << 0.02). Oocytes fertilizing with subsequentfragmentation or degeneration showed progesterone levels significantlyhigher than oocytes fertilizing normaly (P << 0.05). Polyspermicoocytes ( n = 3 ) were associated with very high levels of progesteroneproduction but were not significantiy different due to the lownumbers. Oestradial production per unit protein was significantlygreater in oocytes which degenerated (P << 0.05). TheProtein content of cumuli whose oocytes fertilized appearedto be significantly lower than those which did not (P <<0.05). these results probably reflect the maturity of the folliclealthough direct actions of cumulus products upon gametes cannotbe ruled out.     

Cumulus cells are required for the increased apoptotic potential in oocytes of aged mice

Recent studies with female ICR mice have suggested that oocyte DNA fragmentation is one reason for poor oocyte quality and lower fertility associated with ageing. Since it was not determined if this increased 'apoptotic' potential in aged oocytes is due to changes within the oocyte itself or within the microenvironment of cumulus cells (CC) surrounding the germ cell, we sought to clarify if CC were required to affect the rate of apoptosis in oocytes maintained in vitro. Intact cumulus-oocyte complexes (COC) were retrieved by superovulation of virgin female ICR mice at 7 weeks ('young') or 34-35 weeks ('aged') of age. One-half of the COC in each group were incubated at 37 degrees C in human tubal fluid medium under paraffin oil for 24 h. The other half of the COC in each group were denuded of CC and incubated under the same conditions (denuded oocytes; DO). Following incubation, COC were stripped of adherent CC by gentle pipetting. All DO were then fixed and checked by light microscopy for morphological changes characteristic of apoptosis. In young mice, the presence of CC had no significant effect on oocyte death rate (18 +/- 9% and 14 +/- 6% apoptotic oocytes in COC and DO, respectively; P > 0.05). However, in aged mice the percentage of CC-enclosed oocytes that underwent apoptosis was significantly greater as compared to the death rate in DO (48 +/- 3% versus 19 +/- 8% apoptotic oocytes, respectively; P < 0.05). This increased death potential was due to the presence of CC since the occurrence of apoptosis in DO of aged versus young mice was not significantly different (19 +/- 8% versus 14 +/- 6% apoptotic oocytes, respectively; P > 0.05). These results demonstrate that the age-dependent acceleration of apoptosis in oocytes maintained in vitro requires the CC.