Derivation of human embryonic stem cell line MUSIe001-A from an embryo with homozygous α0-thalassemia (SEA deletion).

MUSIe001-A cell line was derived from a Southeast Asian (SEA) type deletion α0-thalassemia embryo. The SEA deletion embryo was donated for research with informed consent. This cell line shows normal hESC morphology, expresses all pluripotent markers, and has the potential to differentiate into all three germ layers in vitro and in vivo. The MUSIe001-A line has normal karyotype and is free from mycoplasma contamination. PCR analysis confirmed the MUSIe001-A cell line to be a SEA type deletion. MUSIe001-A is a valuable proof of principle model for gene therapy that will facilitate the development of new treatments for affected foetuses.

High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation.

BACKGROUND: Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used. METHODS: We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells. RESULTS: This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen-thawed embryos with better derivation efficiency of more than 50% (range 50-100%; median 70%). CONCLUSIONS: We successfully developed a better hESC derivation methodology using the "MTP" culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.

The correlation of cumulus mucification patterns with oocyte maturation rate in vitro in FSH + LH-primed IVM cycles: a prospective study.

PURPOSE: This prospective study was conducted to compare the oocyte maturation rate in vitro among four types of cumulus cell (CC) morphology, in dual gonadotropin-primed in vitro maturation (IVM) oocyte cycles. METHODS: Two-hundred and thirty cumulus-oocyte complexes (COCs) were retrieved from FSH + hCG-primed in vitro maturation cycles of 20 patients diagnosed with polycystic ovarian syndrome. The COCs that contained immature oocytes were classified into four groups according to their cumulus mucification patterns (dispersed, clumped, compacted or sparse) and their oocyte maturation rates were compared. Chi square test and Fisher's exact test were used as appropriate. RESULTS: Oocytes enclosed by dispersed and clumped CCs exhibited higher maturation rates than those with compacted and sparse patterns (P < 0.001). CONCLUSION: In dual gonadotropin-primed IVM cycles, oocyte maturation rates are highest in those showing dispersed and clumped CC patterns.

Lack of association of rs3798220 with small apolipoprotein(a) isoforms and high lipoprotein(a) levels in East and Southeast Asians.

OBJECTIVE: The variant allele of rs3798220 in the apolipoprotein(a) gene (LPA) is used to assess the risk for coronary artery disease (CAD) in Europeans, where it is associated with short alleles of the Kringle IV-2 (KIV-2) copy number variation (CNV) and high lipoprotein(a) (Lp(a)) concentrations. No association of rs3798220 with CAD was detected in a GWAS of East Asians. Our study investigated the association of rs3798220 with Lp(a) concentrations and KIV-2 CNV size in non-European populations to explain the missing association of the variant with CAD in Asians. METHODS: We screened three populations from Africa and seven from Asia by TaqMan Assay for rs3798220 and determined KIV-2 CNV sizes of LPA alleles by pulsed-field gel electrophoresis (PFGE). Additionally, CAD cases from India were analysed. To investigate the phylogenetic origin of rs3798220, 40 LPA alleles from Chinese individuals were separated by PFGE and haplotyped for further SNPs. RESULTS: The variant was not found in Africans. Allele frequencies in East and Southeast Asians ranged from 2.9% to 11.6%, and were very low (0.15%) in CAD cases and controls from India. The variant was neither associated with short KIV-2 CNV alleles nor elevated Lp(a) concentrations in Asians. CONCLUSION: Our study shows that rs3798220 is no marker for short KIV-2 CNV alleles and high Lp(a) in East and Southeast Asians, although the haplotype background is shared with Europeans. It appears unlikely that this SNP confers atherogenic potential on its own. Furthermore, this SNP does not explain Lp(a) attributed risk for CAD in Asian Indians.

Outcome of sperm preparation using double-gradients technique study in Siriraj Hospital.

OBJECTIVE: To determine the succession of sperm preparation using the double-gradients technique on sperm quality: sperm recovery rate, sperm concentration, sperm motility and percentage of post wash total motile sperm count. STUDY DESIGN: Retrospective descriptive study. SETTINGS: Infertility clinic, Faculty of Medicine, Siriraj hospital. MATERIAL AND METHOD: During the period of January 1, 2002 through December 31, 2007, data including semen analysis before and after IUI procedure were reviewed in all male patients who were referred to the andrology laboratory for sperm washing and IUI. Comparison of semen parameters such as total sperm concentration, total motile sperm count before and after sperm preparation as well as total sperm recovery rate and total motile sperm recovery rate was evaluated. RESULTS: After sperm preparation, both sperm concentration and progressive sperm motility significantly increased, while total motile sperm count significantly decreased. Moreover, the percentage of motile sperm recovery rate and total sperm recovery rate was higher after sperm preparation at around 59.88 +/- 19.26% and 34.03 +/- 14.58% respectively. When categorizing semen parameters to 4 groups: normozoospermia, oligozoospermia, astenozo-ospermia and oligo-astenozoospermia, sperm motility in each group, comparing with sperm motility prior preparation, significantly improved after sperm preparation. Furthermore, motile sperm recovery rate in each group significantly increased except for astenozoospermia. Total sperm recovery rate in oligozoospermia was significantly higher than normozoospermia, yet the others were significantly lower. CONCLUSION: Sperm preparation using double gradient percoll provided a high percentage of motile sperm recovery rate and total sperm recovery rate. It also dramatically improved progressive sperm motility in normozoospermia, oligozoospermia, astenozoospermia and oligo-astenozoospermia.

Vitrification of mouse oocyte using open pulled straws compared with needles.

OBJECTIVE: To compare the survival rate of mouse oocytes and fertilization rate between using open pulled straws (OPS) and needles for vitrification. MATERIAL AND METHOD: Meiosis II oocytes from female C57B/6J mice aged 7-8 weeks were collected and allocated to two groups for vitrification by using OPS or needles. Vitrified oocytes were thawed, morphological survival and fertilization rate were examined. RESULTS: There was no obvious difference between the morphological survival rates of vitrified mouse oocytes using OPS and needles (66.7% vs 64.8%). Proportions Difference 1.9% (95% CI -7.1, 10.7). The vitrified oocytes from the needle had significantly higher percentages of fertilization rate than OPS (76.8% vs 62.5%). Proportions Difference -14.3% (95% CI -24.5, -3.6). CONCLUSION: Vitrification method of mouse oocytes using needles when compared to OPS provides a similar morphological survival rate and higher fertilization rate.

Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization.

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.