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 stem cell research; networking and collaboration.

Stem cell research has obtained more attention during the last decade because of its strong potential as a new tool to cure many chronic diseases. In addition, stem cell knowledge is an important basis for understanding pathophysiology at the cellular level and developing disease models for experimental research. There are different limitations on resources, budget, policy and regulation among countries. As a result, each country has particular advantages and disadvantages in stem cell research. This result in the establishment of international networks and collaborations to coordinate and promote stem cell research aimed at medical applications.

Introduction to stem cell medicine.

Embryonic stem cell is the promising novel therapeutic tool for various degenerative diseases and tissue injuries. With the concept of cell and tissue therapy, many chronic disorders will be curable. The present article provides basic knowledge of stem cell in areas of definition, classification and future clinical applications. In addition, stem cell application is not only focusing on regenerative purpose, but also concentrating on more understanding about the early human development and the pathophysiology of genetic diseases at the cellular level. However, there are some technical problems and ethical concern that should be resolved before applying stem cells into clinical practice.

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 determinant factors and the anxiety level of infertile couples during the treatment of in vitro fertilization and embryo transfer at Chulalongkorn Hospital.

The determinant factors and the anxiety level of infertile couples during the treatment of in vitro fertilization and embryo transfer were studied in 60 infertile couples between 1 January to 31 May 2000. The instruments employed in the study were Personal and Health Data Questionnaire, the Cornell Medical Index, and the Determinant Factors of Anxiety. The average age was between 36-40 years old, holding a Bachelor's degree and working in private companies earning a monthly income between 10,000-20,000 Baht. Most infertile couples wanted to have a child in order to fulfill the meaning of being a "family" and were anxious about the treatment. The couples in general did not have any background of emotional disturbance. Women were found to have a slightly higher anxiety than men. The determinant factors of anxiety were found to be the side-effects of the infertility treatment, inadequate time to consult with the physician/nurse, the outcome of the infertility treatment, possibility to possibility of not succeeding/infertility cannot be treated and the process of the diagnostic procedures accordingly. The results of the study will serve as a guideline for improving better services and understanding between the physician and the patient regarding the expectations of the IVF treatment.

Preliminary study on somatic cell nuclear transfer in rabbits in Thailand.

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.

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.