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EMB-001 Comparative analysis of IVF outcomes with intracytoplasmic sperm injection using ejaculated versus testicular spermatozoa
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were detected by the FACS. The gene profile between the embryonic carcinoma cells and embryonic stem cells were compared by the affymetrix gene chip. The analysis of signal pathways in different genes was performed by the Dchip2004 software. The results of the gene chips in the experiments were verified by real-time PCR. Results: The NTERA-2 cells presented expressed the special markers of stem cells, such as Oct-4, Nanog, Sox-2, TERF1, TERF2, REX, FGF4, Cripto, Thy1, LEFTYA and had alkaline phosphatase and telomerase activity. and it could generate cell aggregates similar to embryoid bodies in embryonic stem cells. The NTERA-2 cells can be induced into three germ layers in vitro and in vivo. The more disordered tissue structures existed in the tumour formed by NTERA-2 cells in vivo compared with human embryonic stem cells. The results of the cell cycle analysis showed that the NTERA-2 cells of S phase (63.1 ± 1.27%) was more than the HES cells (35.1 ± 1.4%). The analysis of apoptosis showed the NTERA-2 cells had stronger anti-apoptosis ability. The microarray analysis and real-time PCR results showed that some oncogenes were up-regulated in the NTERA-2 cells, whereas the genes related to differentiation were down-regulated, and that Wnt signal pathway was activated. Conclusions: The NTERA-2 cells had characteristics of stem cells and malignant tumour during expansion and differentiation, In the sense, the NTERA-2 cells were one type of cancer stem cell. The difference between human embryonic stem cells and embryonal carcinoma cells (NTERA-2) may provide a valuable insight into cancer stem cells derived from the embryonic phase. ASCR-016 Comparison of the effect of soluble TRAIL from progression-elevated gene-3 promoter on EC and ES cells Jiang X, Xia H, Du L, Yang S, Lu G Institute of Reproduction and Stem Cell Engineering, China [email protected] Introduction: Pluripotent embryonal carcinoma (EC) cells derived from teratocarcinomas that arise from transformed germ cells are generally considered to be the malignant counterparts of human embryonic stem cells. Thus, EC cells are regarded as a mode for study of cancer stem cells. The soluble TRAIL (sTRAIL) driven by progression-elevated gene-3 promoter can induce the apoptosis of EC cells whereas its effect on embryonic stem (ES) cells still remains unknown. Materials/Methods: A vector expressing tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL amino acids 114–281) driven by progression-elevated gene3 promoter (PEG-prom) was constructed. The EC and ES cells were transfected with the AAV-PEG-sTRAIL vector respectively. The cell apoptosis were evaluated by FACS and the expression of mRNA level of DcR1, DcR2, DR4 and DR5 with real-time PCR. Results: EC cells demonstrated significant higher apoptosis ration than ES cells with the influence of sTRAIL. Moreover, EC cells showed about (>2-fold) increase in DR5 expression whereas expression no significant change was found in the expression of DcR1, DcR2 and DR4. However, all of these genes showed no significant change in ES cells. Conclusion: Our results indicate that sTRAIL plays specific role in EC cells rather than ES cells, which provides a potential method for gene therapy of cancer stem cells.
EMB-001 Comparative analysis of IVF outcomes with intracytoplasmic sperm injection using ejaculated versus testicular spermatozoa Majumdar G, Gaur R, Mittal S, Majumdar A Centre of IVF and Human Reproduction, Sir Ganga Ram Hospital, New Delhi, India [email protected] Introduction: Intracytoplasmic sperm injection (ICSI) has become the treatment of choice for infertile couples with subnormal semen parameters. Our objective was to determine if varying semen characteristics and type of spermatozoa alter IVF outcome in patients undergoing ICSI. Materials/Methods: In a retrospective analysis, 262 patients undergoing ICSI between January 2005 and September 2006 were divided into four groups based on sperm characteristics: normozoospermia, oligozoospermia (1–5 × 106/ml), severe oligoasthenozoospermia (OA) (<1 × 106/ml) and testicular spermatozoa. The main outcome measures were fertilization rate, clinical pregnancy rate per embryo transfer and abortion rate. Results: Fertilization rates were similar with ejaculated and testicular spermatozoa (66.0% versus 70.1%) respectively. However, within the ejaculated group, fertilization rate was significantly lower (P < 0.01) in men with severe OA (53.53%) as compared with normal (68.6%) and oligozoospermic men (71.5%). The level of statistical significance was set at 10%. The clinical pregnancy rates in men with normal spermatozoa (31.25%; 40/128), oligozoospermia (29.62%; 16/54), testicular spermatozoa (29.41%; TESA: 8/28, PESA: 2/6) were also statistically higher than men showing severe OA (18.18%; 8/44). There was one case of severe teratozoospermia that resulted in a pregnancy and a live birth. The overall abortion rate was 22.66% (17/75) and differences in pregnancy loss within the groups were not significant. Conclusions: Our analysis showed that fertilization and pregnancy rates were lowest in patients undergoing ICSI with ejaculated spermatozoa showing severe OA. Otherwise fertilization and pregnancy rates were comparable using ejaculated and testicular spermatozoa. It seems necessary to investigate if testicular spermatozoa could make a better choice in patients with severe OA to improve ICSI outcome in such patients. EMB-002 Borealin is specifically expressed in ES cells and involved in mouse embryo development Zhang QJ, Lu GX Institute of Human Reproduction and Stem Cell Engineering, China [email protected] Introduction: Borealin was highly expressed in the undifferentiated embryonic stem cells, the mouse preimplantation embryos and the brain of 8.5–9.5-day postcoitum mouse embryos. Furthermore, due to borealin depletion by microinjecting anti-borealin antibody into the zygotes the mouse embryos were arrested at the 2- or 4-cell stage and their chromosomes could not correctly localize at the equator plane of the mitotic spindle and most cells had two or more nuclei. Taken together, these results indicate that borealin is necessary for HES cell proliferation and plays a crucial role in the early
S-35 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008
were detected by the FACS. The gene profile between the embryonic carcinoma cells and embryonic stem cells were compared by the affymetrix gene chip. The analysis of signal pathways in different genes was performed by the Dchip2004 software. The results of the gene chips in the experiments were verified by real-time PCR. Results: The NTERA-2 cells presented expressed the special markers of stem cells, such as Oct-4, Nanog, Sox-2, TERF1, TERF2, REX, FGF4, Cripto, Thy1, LEFTYA and had alkaline phosphatase and telomerase activity. and it could generate cell aggregates similar to embryoid bodies in embryonic stem cells. The NTERA-2 cells can be induced into three germ layers in vitro and in vivo. The more disordered tissue structures existed in the tumour formed by NTERA-2 cells in vivo compared with human embryonic stem cells. The results of the cell cycle analysis showed that the NTERA-2 cells of S phase (63.1 ± 1.27%) was more than the HES cells (35.1 ± 1.4%). The analysis of apoptosis showed the NTERA-2 cells had stronger anti-apoptosis ability. The microarray analysis and real-time PCR results showed that some oncogenes were up-regulated in the NTERA-2 cells, whereas the genes related to differentiation were down-regulated, and that Wnt signal pathway was activated. Conclusions: The NTERA-2 cells had characteristics of stem cells and malignant tumour during expansion and differentiation, In the sense, the NTERA-2 cells were one type of cancer stem cell. The difference between human embryonic stem cells and embryonal carcinoma cells (NTERA-2) may provide a valuable insight into cancer stem cells derived from the embryonic phase. ASCR-016 Comparison of the effect of soluble TRAIL from progression-elevated gene-3 promoter on EC and ES cells Jiang X, Xia H, Du L, Yang S, Lu G Institute of Reproduction and Stem Cell Engineering, China [email protected] Introduction: Pluripotent embryonal carcinoma (EC) cells derived from teratocarcinomas that arise from transformed germ cells are generally considered to be the malignant counterparts of human embryonic stem cells. Thus, EC cells are regarded as a mode for study of cancer stem cells. The soluble TRAIL (sTRAIL) driven by progression-elevated gene-3 promoter can induce the apoptosis of EC cells whereas its effect on embryonic stem (ES) cells still remains unknown. Materials/Methods: A vector expressing tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL amino acids 114–281) driven by progression-elevated gene3 promoter (PEG-prom) was constructed. The EC and ES cells were transfected with the AAV-PEG-sTRAIL vector respectively. The cell apoptosis were evaluated by FACS and the expression of mRNA level of DcR1, DcR2, DR4 and DR5 with real-time PCR. Results: EC cells demonstrated significant higher apoptosis ration than ES cells with the influence of sTRAIL. Moreover, EC cells showed about (>2-fold) increase in DR5 expression whereas expression no significant change was found in the expression of DcR1, DcR2 and DR4. However, all of these genes showed no significant change in ES cells. Conclusion: Our results indicate that sTRAIL plays specific role in EC cells rather than ES cells, which provides a potential method for gene therapy of cancer stem cells.
EMB-001 Comparative analysis of IVF outcomes with intracytoplasmic sperm injection using ejaculated versus testicular spermatozoa Majumdar G, Gaur R, Mittal S, Majumdar A Centre of IVF and Human Reproduction, Sir Ganga Ram Hospital, New Delhi, India [email protected] Introduction: Intracytoplasmic sperm injection (ICSI) has become the treatment of choice for infertile couples with subnormal semen parameters. Our objective was to determine if varying semen characteristics and type of spermatozoa alter IVF outcome in patients undergoing ICSI. Materials/Methods: In a retrospective analysis, 262 patients undergoing ICSI between January 2005 and September 2006 were divided into four groups based on sperm characteristics: normozoospermia, oligozoospermia (1–5 × 106/ml), severe oligoasthenozoospermia (OA) (<1 × 106/ml) and testicular spermatozoa. The main outcome measures were fertilization rate, clinical pregnancy rate per embryo transfer and abortion rate. Results: Fertilization rates were similar with ejaculated and testicular spermatozoa (66.0% versus 70.1%) respectively. However, within the ejaculated group, fertilization rate was significantly lower (P < 0.01) in men with severe OA (53.53%) as compared with normal (68.6%) and oligozoospermic men (71.5%). The level of statistical significance was set at 10%. The clinical pregnancy rates in men with normal spermatozoa (31.25%; 40/128), oligozoospermia (29.62%; 16/54), testicular spermatozoa (29.41%; TESA: 8/28, PESA: 2/6) were also statistically higher than men showing severe OA (18.18%; 8/44). There was one case of severe teratozoospermia that resulted in a pregnancy and a live birth. The overall abortion rate was 22.66% (17/75) and differences in pregnancy loss within the groups were not significant. Conclusions: Our analysis showed that fertilization and pregnancy rates were lowest in patients undergoing ICSI with ejaculated spermatozoa showing severe OA. Otherwise fertilization and pregnancy rates were comparable using ejaculated and testicular spermatozoa. It seems necessary to investigate if testicular spermatozoa could make a better choice in patients with severe OA to improve ICSI outcome in such patients. EMB-002 Borealin is specifically expressed in ES cells and involved in mouse embryo development Zhang QJ, Lu GX Institute of Human Reproduction and Stem Cell Engineering, China [email protected] Introduction: Borealin was highly expressed in the undifferentiated embryonic stem cells, the mouse preimplantation embryos and the brain of 8.5–9.5-day postcoitum mouse embryos. Furthermore, due to borealin depletion by microinjecting anti-borealin antibody into the zygotes the mouse embryos were arrested at the 2- or 4-cell stage and their chromosomes could not correctly localize at the equator plane of the mitotic spindle and most cells had two or more nuclei. Taken together, these results indicate that borealin is necessary for HES cell proliferation and plays a crucial role in the early
S-35 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008