Search

Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line.

Purpose: To determine the correlation between the follicular sizes and oocyte recovery, metaphase II oocyte recovery, fertilization rate and good embryo quality from mature and immature oocytes in an intracytoplasmic sperm injection (ICSI) program. Methods: 991 follicles obtained from 72 ICSI cycles were classified into three groups according to their diameters as measured by transvaginal ultrasound including group A ( 14 mm). All obtained oocytes were classified according to their nuclear maturation: germinal vesicle (GV), metaphase I (MI) and metaphase II (MII). Mature oocytes underwent ICSI while immature oocytes were further cultured until maturity before ICSI was performed. The rates of fertilization and good quality embryos at day 3 were evaluated. Results: A progressive and significant increase in the rates of oocyte recovery and MII oocyte recovery were observed from group A follicles compared to the other groups (p < 0.001). The fertilization rate of mature and in vitro matured oocytes, as well as the rate of good quality embryos showed a tendency to increase from group A to group C follicles, but not significantly. The corresponding fertilization rates were 78 and 55.3% (p < 0.001) for mature and in vitro matured oocytes, respectively. Conclusion: Collection of oocytes from small follicles, especially with a mean diameter less than 10 mm, and in vitro maturation of immature oocytes before fertilization may allow the total number of good quality and transferable embryos to be increased.

We have shown that sperm sulfolipidimmobilizing protein 1 (SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid (SGG)-binding protein, is significant in sperm-zona pellucida (ZP) interaction. The objective of this study was to localize SLIP1 on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1 Fab before coincubation with sperm, showed a significant, dose-dependent decrease in sperm-egg plasma membrane binding. Similar results were obtained when affinity-purified antiSLIP1 IgG was used for egg pretreatment. The significance of egg SLIP1 in sperm-egg plasma membrane binding was further demonstrated by a decrease (36-52%) in in vitro fertilization when zona-intact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has been shown to bind SGG in vitro, we investigated the possibility that sperm SGG may participate in sperm-egg plasma membrane binding through egg SLIP1. Pretreatment of sperm with antiSGG Fab prior to coincubation with zona-free eggs resulted in a dose-dependent decrease in sperm-egg plasma membrane binding. Collectively, these findings strongly suggest a role for egg SLIP1 in sperm-egg plasma membrane interaction, which may be through its binding to sperm SGG.

Purpose:Pregnancy and implantation rates after mechanical assisted hatching (AH) in patients aged ≥38 years, with embryos ≥15 μm in zonal thickness and two or more failed attempts, were assessed at two infertility centers using fresh and frozen embryo transfer (FET) cycles. Methods:AH was performed on 3-day-old embryos. Spare embryos cryopreserved at the two-pronucleus stage were subjected to AH after 2 days of culture and transferred to artificially prepared uteri. Results:In fresh cycles, no significant differences in pregnancy rates (clinical and ongoing) and implantation rates were observed between the AH and the controls for all three selected patient groups (Centers 1 and 2). In FET cycles, AH tended to give poor results for ≥38 year olds (clinical pregnancy rates of 0 and 5.0% with AH vs 13.3 and 16.7% for controls at Centers 1 and 2, respectively). With AH, embryos with thick zonae implanted to the same extent as those in the control group and achieved pregnancies for patients with multiple failures (four to six attempts for some) in both fresh and FET cycles. Conclusions:AH failed to show significant benefits in all three patient groups. A larger study group may confirm the effects of AH on frozen/thawed embryos and outcomes for multiple failure cases.

Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 μM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation.

Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research.

To assess the embryo formation rate after electrical activation on failed-to-fertilize oocytes after intracytoplasmic sperm injection (ICSI).A prospective, randomized, experimental study was conducted in our research laboratory at the Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, between January and September 2004. One hundred failed-to-fertilize oocytes after ICSI were randomly assigned by stratified allocation according to oocyte grading before ICSI. Fifty unfertilized oocytes were activated with 3 consecutive 40-/microsec pulses of 1.5 kV/cm direct electrical current in buffer solution. The other 50 unfertilized oocytes, composing the control group, were treated in same way but without electrical activation. The embryo formation rates between the groups were compared.Embryo formation rates in the electrically activated group were 80% as compared with 16% in the control group (p0.001).Failed-to-fertilize oocytes after ICSI seem to be able to resume embryonic development after electrical activation.

The objective of the study was to develop the somatic nuclear transfer technique by using rabbits as the model. The oocyte recipients aged 16 h post coitus were collected surgically from 20 superovulated rabbit doe with 28 and 40 mg Follicle Stimulating Hormone (FSH) after mating with a vasectomized male. The metaphase II plate and 1st polar body of oocyte was later aspirated by enucleated micropipette under an inverted microscope. A single donor cell; cumulus cell or cultured or frozen fibroblast cell from passage 1 to 9 were transferred to enucleated oocyte and fused with triple DC pulses, 3.2 kv, 20 micros. The fused embryos were cultivated in TCM 199 NaHCO3 + 10 per cent fetal calf serum (FCS) for 4 days. The cleavage rate (2-cell stage) was 37.2 per cent (32/86) from eight experiments, and 18.8 per cent (6/32) developed to the early morula stage. This study also indicated that the enucleation pipette and the somatic cell type influenced the success.

To investigate the effect of growth hormone on the development of in vitro matured unstimulated human oocytes.Randomized controlled study.Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn university.108 germinal vesicle-stage oocytes were retrieved from 47 patients undergoing gynecologic surgery. They were aspirated either during gynecologic surgery or from excised ovaries. The oocytes were then cultured in vitro with or without growth hormone (1,000 ng/ml) in medium199 supplemented with sodium pyruvate, FSH, LH, antibiotic and synthetic serum. Incubation was done at 37 degree C with 5 per cent CO2 in air and nuclear stage was assessed after 18, 42, 66 and 90 h of incubation.Attainment of metaphase II and GVBD RESULTS: After in vitro culture, there were no significant differences in maturation and GVBD rate. 27 of 52 (51.9%) oocytes (GV) in growth hormone group matured to metaphase II compared with 25 of 53 (47.2%) GV in control group. GVBD rate for germinal vesicle-stage in growth hormone group was 76.9 per cent compared with 79.2 per cent in control group.Culture of immature oocytes in vitro with growth hormone results in similar maturation rate as that without GH.