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Impact of Apoptosis on Sperm Morphology Indices
REPRODUCTIVE BIOLOGY: HUMAN STUDIES P-698 Impact of Apoptosis on Sperm Morphology Indices. N. Aziz, T. M. Said, U. Paasch, S. Grunewald, H. Glander, A. Agarwal. Liverpool Women’s Hospital, Liverpool, United Kingdom; Cleveland Clinic Foundation, Cleveland, OH; Department of Dermatology/Andrology, Leipzig University, Leipzig, Germany. OBJECTIVE: Apoptosis has been implicated as a cause of male infertility. Externalization of phosphatidylserine (PS) to the sperm outer membrane leaflet is considered to mark terminal apoptosis. Magnetic cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads eliminates spermatozoa with externalized PS. The procedure delivers 2 sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). The relationship between apoptosis and the sperm morphology has not been well characterized. Sperm deformity index (SDI) is a novel quantitative expression of sperm morphological quality that has an enhanced predictive power and reproducibility. The aim of this study was to investigate the relationship between apoptosis and the sperm morphology profile including the SDI score. DESIGN: Prospective study. MATERIALS AND METHODS: Semen specimens collected from 15 healthy donors were prepared by density gradient centrifugation followed by MACS using annexin V-conjugated microbeads. Sperm morphology was assessed using the WHO guidelines (1999) and the Tygerberg’s strict criteria. The SDI score was calculated by dividing the total number of deformities observed by the number of sperm randomly selected and evaluated, irrespective of their morphological normality. Apoptosis was evaluated in spermatozoa using flow cytometry coupled with fluorochrome stains that detect: caspase-3 activation (CP3), disruption of mitochondrial potential (MMP) and externalization of PS. Results are expressed as mean ⫾ standard deviation. Pair-wise comparisons were made using Student’s t-test and correlation coefficients were calculated by Spearman’s rank test. RESULTS: The mean of percentage normal sperm morphology and the mean SDI scores in the annexin-negative and the annexin-positive fractions are shown in the table below. The annexin-negative fraction separated by MACS had significantly lower mean SDI score compared to the annexinpositive fraction (p⬍0.0001). On the other hand, the mean percentage of normal sperm morphology using both WHO and the strict Tygerberg’s criteria were comparable between the 2 fractions. SDI scores showed significant positive correlation with CP-3 activation (r⫽0.68, p⬍.0001), MMP integrity (r⫽-0.55, p⫽0.002) and externalization of PS (r⫽0.72, p⬍0.0001). There was no evidence of a significant correlation between normal sperm morphology using both WHO and the strict Tygerberg’s criteria and the three apoptotic markers. CONCLUSION: Apoptosis shows significantly positive correlation with sperm morphology using the SDI scores. Non-apoptotic spermatozoa show lower incidence of morphological anomalies. The SDI can detect subtle differences in sperm morphology that may pass undetected using routine assessment of normal sperm morphology. Supported by: None
OBJECTIVE: Akt is activated via phosphatidylinositol 3-kinase (PI3kinase) by several growth factors and plays various important roles in, such as in cell growth, apoptosis, glycogen synthesis, cell migration and invasiveness. By analyzing profiles of differential gene expressions using a cDNA array system before and after implantation of a mouse embryo, we identified phosphorylated Akt (pAkt) as a gene associated with decidualization and apoptosis in mouse endometrium. In the present study, we investigated whether pAkt is expressed in normally cycling human endometrium and decidua, and whether IGF-1 is involved in the phosphorylation of Akt in these tissues. DESIGN: Descriptive study of the expression of phosphorylated protein and controlled in vitro experiments. MATERIALS AND METHODS: Endometrial samples were taken from hysterectomy or endometrial biopsy specimens from 34 pre-menopausal non-pregnant women after obtaining written consent. The decidual tissues were obtained from 6 ectopic pregnancies. Endometrial samples were dated using Noyes’ histological criteria. Tissue fragments for protein extraction were flash frozen in liquid nitrogen. Tissues for immunohistochemistry were fixed in 4% paraformaldehyde and embedded in paraffin. Western blot analysis was performed with antibodies against Akt(1: 1000) or PhosphoAkt(Ser 473)(1: 1000). Immunohistochemistry was performed using polyclonal antibody for phospho-Akt (Ser 473). Endometrial stromal cells were separated from late proliferative endometrium through collagenase treatment and mesh-filtration. The isolated endometrial stromal cells were cultured in DMEM/F12 medium supplemented with 10% charcoal-dextranstripped FBS for 7 days, and were pretreated with E2 (10-9mol/l) and P4 (10-7mol/l) every 2 days for 7 days. After pre-treatment, cells were cultured in the serum-free medium for 24 h before treatment with IGF-1 with and without LY294002, inhibitors of phosphatidylinositol 3-kinase. RESULTS: Though the level of Akt protein is equivalent through the menstrual cycle, the level of pAkt(Ser473) protein is particularly increased in proliferative, late secretory endometrium, and decidual tissues. Immunohistochemistry showed that the expression of pAkt was localized in glandular epithelial cells of basal and functional layers, and in stromal cells of the functional layer. There was no expression of pAkt in stromal cells of the basal layer. The expression of pAkt was in proportion to the decidual reaction. In decidual tissue, decidual cells exhibited marked staining of the cytoplasm and nuclei. In cultured cells treated with E2 and P4, the production of PRL mRNA was confirmed. The expression of Akt had no obvious difference with hormone treatment, whereas that of pAkt was markedly decreased by it. Expression of pAkt was recovered by administration of IGF-1 in hormone-treated cultured cells. Phosphorylation of Akt by IGF-1 was blocked by LY294002. CONCLUSION: pAkt was strongly expressed in decidual tissues in vivo, while pAkt was not up-regulated with sex steroid treatment, unless with IGF-1 treatment in cultured stromal cells. Our findings in vivo and in vitro showed that the phosphorylation of Akt can be controlled by IGF-1 as well as sex steroid hormones in human endometrial tissues, and may play an important role in early pregnancy. Supported by: Bio-oriented Technology Research Advancement Institution (BRAIN)
P-700 Table. Mean percentage of normal sperm morphology and mean SDI scores in the annexin-negative and the annexin-positive fractions.
P-699 Insulin-Like Growth Factor-I Regulates Phosphorylation of Akt in Human Endometrial Stromal Cells. T. Hara, A. Toyofuku, Y. Kudo. Hiroshima University, Faculty of Medicine, Hiroshima, Japan.
FERTILITY & STERILITY威
Evidence of Transforming Growth Factor -2 Production in Culture Media by Human Embryos. M. A. Bedaiwy, T. Falcone, T. M. Said, S. Worley, J. Thornton, A. Agarwal. Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The biological, biochemical, and metabolic properties of pre-implantation embryos are not well understood. Recently, many growth factors have been implicated in folliculogenesis. Whether this involvement extends to the ex vivo life is not yet established. TGF- family is a known cell-to-cell communication molecule. We have shown that TGF-1 is produced by human embryos in vitro. The objective of this study was to characterize and examine the relationship of early human embryonic development parameters with day 1 culture media TGF-2 levels. DESIGN: Prospective study MATERIALS AND METHODS: Patients undergoing in vitro fertilization (IVF; 13 with intracytoplasmic sperm injection (ICSI) and 18 without ICSI in 31 cycles) were included. Fertilization and early culture were performed in HTF with 5% serum substitute supplement. D-1 TGF 2 levels in the central well (sample) and the outer well (control) of each
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OBJECTIVE: Akt is activated via phosphatidylinositol 3-kinase (PI3kinase) by several growth factors and plays various important roles in, such as in cell growth, apoptosis, glycogen synthesis, cell migration and invasiveness. By analyzing profiles of differential gene expressions using a cDNA array system before and after implantation of a mouse embryo, we identified phosphorylated Akt (pAkt) as a gene associated with decidualization and apoptosis in mouse endometrium. In the present study, we investigated whether pAkt is expressed in normally cycling human endometrium and decidua, and whether IGF-1 is involved in the phosphorylation of Akt in these tissues. DESIGN: Descriptive study of the expression of phosphorylated protein and controlled in vitro experiments. MATERIALS AND METHODS: Endometrial samples were taken from hysterectomy or endometrial biopsy specimens from 34 pre-menopausal non-pregnant women after obtaining written consent. The decidual tissues were obtained from 6 ectopic pregnancies. Endometrial samples were dated using Noyes’ histological criteria. Tissue fragments for protein extraction were flash frozen in liquid nitrogen. Tissues for immunohistochemistry were fixed in 4% paraformaldehyde and embedded in paraffin. Western blot analysis was performed with antibodies against Akt(1: 1000) or PhosphoAkt(Ser 473)(1: 1000). Immunohistochemistry was performed using polyclonal antibody for phospho-Akt (Ser 473). Endometrial stromal cells were separated from late proliferative endometrium through collagenase treatment and mesh-filtration. The isolated endometrial stromal cells were cultured in DMEM/F12 medium supplemented with 10% charcoal-dextranstripped FBS for 7 days, and were pretreated with E2 (10-9mol/l) and P4 (10-7mol/l) every 2 days for 7 days. After pre-treatment, cells were cultured in the serum-free medium for 24 h before treatment with IGF-1 with and without LY294002, inhibitors of phosphatidylinositol 3-kinase. RESULTS: Though the level of Akt protein is equivalent through the menstrual cycle, the level of pAkt(Ser473) protein is particularly increased in proliferative, late secretory endometrium, and decidual tissues. Immunohistochemistry showed that the expression of pAkt was localized in glandular epithelial cells of basal and functional layers, and in stromal cells of the functional layer. There was no expression of pAkt in stromal cells of the basal layer. The expression of pAkt was in proportion to the decidual reaction. In decidual tissue, decidual cells exhibited marked staining of the cytoplasm and nuclei. In cultured cells treated with E2 and P4, the production of PRL mRNA was confirmed. The expression of Akt had no obvious difference with hormone treatment, whereas that of pAkt was markedly decreased by it. Expression of pAkt was recovered by administration of IGF-1 in hormone-treated cultured cells. Phosphorylation of Akt by IGF-1 was blocked by LY294002. CONCLUSION: pAkt was strongly expressed in decidual tissues in vivo, while pAkt was not up-regulated with sex steroid treatment, unless with IGF-1 treatment in cultured stromal cells. Our findings in vivo and in vitro showed that the phosphorylation of Akt can be controlled by IGF-1 as well as sex steroid hormones in human endometrial tissues, and may play an important role in early pregnancy. Supported by: Bio-oriented Technology Research Advancement Institution (BRAIN)
P-700 Table. Mean percentage of normal sperm morphology and mean SDI scores in the annexin-negative and the annexin-positive fractions.
P-699 Insulin-Like Growth Factor-I Regulates Phosphorylation of Akt in Human Endometrial Stromal Cells. T. Hara, A. Toyofuku, Y. Kudo. Hiroshima University, Faculty of Medicine, Hiroshima, Japan.
FERTILITY & STERILITY威
Evidence of Transforming Growth Factor -2 Production in Culture Media by Human Embryos. M. A. Bedaiwy, T. Falcone, T. M. Said, S. Worley, J. Thornton, A. Agarwal. Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The biological, biochemical, and metabolic properties of pre-implantation embryos are not well understood. Recently, many growth factors have been implicated in folliculogenesis. Whether this involvement extends to the ex vivo life is not yet established. TGF- family is a known cell-to-cell communication molecule. We have shown that TGF-1 is produced by human embryos in vitro. The objective of this study was to characterize and examine the relationship of early human embryonic development parameters with day 1 culture media TGF-2 levels. DESIGN: Prospective study MATERIALS AND METHODS: Patients undergoing in vitro fertilization (IVF; 13 with intracytoplasmic sperm injection (ICSI) and 18 without ICSI in 31 cycles) were included. Fertilization and early culture were performed in HTF with 5% serum substitute supplement. D-1 TGF 2 levels in the central well (sample) and the outer well (control) of each
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