Development of human embryonic stem cell derivation.

OBJECTIVE: To establish human embryonic stem (hES) cells from human embryos. DESIGN: Experimental study. SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University. MATERIAL AND METHOD: Abnormal and normal fertilization embryos were cultured in vitro until reaching blastocyst stage. Four different methods for isolation of ICMs were used. Immunosurgery, mechanical isolation, laser assists, and whole blastocyst culture were performed. The feeder layers used in the present study were fibroblasts, isolated from either mouse or human. Mechanical splitting of ICM outgrowths or hES-like cells was performed for propagation of cells. Characterization of hES-like cells was conducted by morphology, detection of immunostaining of Oct-4, and enzymatic activity of alkaline phosphatase (AP). HES-like cells were spontaneously differentiated through suspension culture of embryoid body (EB). Subsequent differentiation was done on gelatin-coated dishes. MAIN OUTCOME MEASURE: Establishment of hES cells. RESULTS: By using abnormal fertilization embryos, 80.0% (8/10) of blastocysts were able to attach on the feeder layers, 50% (4/8) formed ICM outgrowths, but no hES-like cells were established. By using normal fertilization embryos, 84.6% (22/26) of blastocysts were able to attach on feeder layers, 18.2% (4/22) formed ICM outgrowths. One hES-like cell line was successfully established by using mechanical isolation of ICMs and human adult skin fibroblasts as feeder layers. This hES-like cells exhibited typical morphology of hES cells, positive staining for Oct-4 and AP. hES-like cells were able to form EB and differentiated into neural-like cells. CONCLUSION: This is the first report in Thailand that hES-like cells can be isolated from normal development human embryos at blastocysts-stage using mechanical isolation of ICM and culture with human adult skin fibroblast as feeder layers.

Effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocysts and subsequent derivation of embryonic stem (ES) cells.

OBJECTIVE: To determine the effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocyst and the efficiency of embryonic stem (ES) cell derivation. DESIGN: Experimental study SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University MATERIAL AND METHOD: In vivo fertilized zygotes were collected and subjected to in vitro culture in potassium simplex optimized medium (KSOM) containing 1,000 unit/ml LIF. The developmental ability of the zygote to blastocyst-stage and the cell numbers in blastocysts were evaluated Expanded blastocysts developed in different culture media were subsequently subjected to ES cell derivation. MAIN OUTCOME MEASURE (s): The influence of LIF on the quality of and the total cell numbers of blastocyst developed in vitro. RESULTS: Supplementation of LIF in KSOM increased the rate of hatching blastocysts (63.8% vs. 53.7%; p < 0.05) and total cell numbers (91.4 +/- 15.0 vs. 85.1 +/- 7.7; p < 0.05) compared to KSOM alone. ES cells were obtained 66.7% from blastocysts developed in KSOM-LIF versus 41.7% in KSOM (p > 0.05). Established ES cell lines showed typical colony and characteristics of pluripotent murine ES cells. CONCLUSION: LIF improved the quality of in vitro produced blastocysts but not enhanced ES cell derivation.

International trends in bioethics for embryonic stem cell research.

Embryonic stem cell (ESC) has been recognized as one of the most promising therapeutic tools for the next decade. However, there are many controversial issues in bioethics for this challenging research area. Each country has its own distinct regulations and policies for ESC research due to their differences in cultural background, religious belief and political influence. These differences will eventually play an important role on international ESC research collaboration. The present article will provide a concise summary of the different policies and regulations regarding bioethical points for ESC research worldwide and show current progress towards establishing standard bioethical guidelines for ESC research on the international level.

Cell therapy: hype or hope.

Cell therapy is a promising therapeutic tool for the next decade. It has a potential to cure a number of chronic diseases and conditions related to aging processes or degenerative changes. In addition, it could be used to replace cells and tissues in injured organs. Furthermore, it may provide a novel approach to congenital anomalies and genetic disorders where current therapeutic options are limited However, many crucial questions need answers to ensure a safe, effective and successful solution in the field of cell therapy. In Thailand, innovative knowledge and expertise in stem cell biology and technology are required as the key elements to make cell therapy a "real" hope.

Comparison of two different fixed doses of follitropin-beta in controlled ovarian hyperstimulation: A prospective randomized, double blind clinical trial.

A prospective randomized, double blind, single centre study was conducted to compare the efficacy, efficiency and clinical side effects of daily fixed dose regimen of either 100 IU or 200 IU of recombinant follicle stimulating hormone(rFSH) Follitropin beta in down-regulated women undergoing controlled ovarian hyperstimulation(COH) for either conventional in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI). A total of sixty women were randomly allocated according to the criteria for the treatment by either 100 IU(n = 30) or 200 IU (n = 30) of FSH. Although more cycle cancellations due to low response were observed in the 100 IU group (n = 9 vs n = 2), two cases of mild and moderate ovarian hyperstimulation syndrome were noted in the higher dose group. Subjects in the group treated with 200 IU appeared to yield more follicles > 17 mm (4.4 vs 3.3, p = 0.05) and more oocytes compared to the group treated with 100 IU (9.2 versus 6.0 oocytes, NS). The total dosage required to develop at least three follicles according to the protocol was significantly lower in the group treated with 100 IU (1203.33 versus 2106. 67, P < 0.0001). In conclusion, a fixed daily dose of 200 IU of rFSH Follitropin beta compared to a fixed daily dose of 100 IU is more effective in terms of follicles > 17 mm development and the number of oocytes retrieved along with a lower cancellation rate, but less efficient as indicated by a higher total rFSH dose needed

The effect of growth hormone on the development of in vitro matured unstimulated human oocytes.

OBJECTIVE: To investigate the effect of growth hormone on the development of in vitro matured unstimulated human oocytes. DESIGN: Randomized controlled study. SETTING: Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn university. MATERIAL AND METHOD: 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. MAIN OUTCOME MEASURE: 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. CONCLUSION: Culture of immature oocytes in vitro with growth hormone results in similar maturation rate as that without GH.

Predictive value of human chorionic gonadotropin in the outcome of early pregnancy achieved by assisted reproductive technology.

This study was undertaken to evaluate the predictive value of the serum human chorionic gonadotropin (hCG) in pregnancies achieved by assisted reproductive techonology (ART). Two hundred and eighty-six pregnancies were studied retrospectively from September 1989 to June 1998. The serum hCG samples at 2-6 weeks after embryo transfer (ET) were analysed by fluoroimmunoassay. Pregnancy status was followed by ultrasonography. There were 100 nonviable pregnancies (NP), 140 viable single pregnancies (VSP) and 46 viable multiple pregnancies (VMP). The sensitivity, specificity, positive and negative predictive value of the D14 hCG (<160 mIU/ml) in distinguishing NP from VSP were 79 per cent, 75 per cent, 68 per cent and 84 per cent, respectively and of the D14 hCG (>350 mIU/ml) in distinguishing VMP from VSP were 82 per cent, 75 per cent, 56 per cent and 91 per cent, respectively. In conclusion, the serum hCG may be used to predict the outcome of early pregnancy achieved by ART.