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ICSI cycles
Abstracts / Journal of Reproductive Immunology 75 (2007) A3–A17 S15 Significance of soluble HLA-G detection in follicular fluids and embryo supernatants in IVF/ICSI cycles N. L´ed´ee a , F. Thonon b , S. Perrier b , J.M. Foidart b , N. Heck b , C. Munault c , R. Lombroso a , J. Selva a , M. Berg`ere a , P. Cavelot a , I. Hammoud a , N. Louafi a , N. Kozma c , M. Aguerre-Girr c , P. Le Bouteiller c , G. Chaouat d , J. Tabiasco c a M´edecine et Biologie de la Reproduction, Centre hospitalier Poissy-st Germain en Laye, Universit´e Versailles St Quentin en Yvelines, Poissy, France; b Departement universitaire de Gynecologie et Obstetrique, CHR Citadelle, Liege, Belgique; c INSERM U563, CHU Purpan, Toulouse, France; d INSERM U781, Clamart, France
Introduction: We set a multicentric blind study to document if soluble (s)HLA-G detection in either individual follicular fluids (FF) or embryo culture supernatants (ES) could be a marker of any potential of clinical pregnancy. Materials and methods: One hundred and eighty-eight individual follicular fluids, 358 Day-2 embryo culture supernatants (Day-2ES) (Center 1) and 595 day-3 embryos supernatants (Day-3 ES) (Center 2) were collected, anonymised and blindly analyzed. Traceability from fully documented embryos was established up to the implantation for 142 FF, 148 Day-2 and 177 Day-3 ES using the Web-based database (Medifirst). The clinical implantation rate of the embryo corresponding to each tested sample was defined as the number of yolk sacs/number of embryos transferred. sHLA-G was blindly detected using an optimized chemioluminescent ELISA assay (Center 3). Threshold of detection was determined in each experiment by the cut-off leading to less than 1 false positive on 60 blanks. The outcomes of the study were: the sHLA-G percentages and medians of detection in FF, Day2 and Day-3 ES; the TOP (4–5 cells on Day-2, 8–9 on Day-3, less than 10% of fragmentation, regular cells) or no TOP (other patterns) embryo repartition; the individual embryo clinical implantation rates (IR) according to the sHLA-G detection in either unpooled FF or singly cultured ES. Results: sHLA-G in FF, D2 and D3 ES were detected in respectively 66% (median: 45 ng/ml), 18.7% (median: 0 ng/ml) and 44% (median: 0 ng/ml) of the samples. FF and Day-2 sHLA-G concentrations were not correlated for the same embryo. TOP and nonTOP embryo repartitions were not influenced by sHLA-G concentrations in FF, Day-2 and Day-3 ES. In the FF, sHLA-G concentrations do not significantly influence the implantation rates even if a tendency to higher implantation rate is observed above 30 ng/ml (8.6% versus 16.9%; p = 0.07). For Day-2 ES, IR was higher in the detectable group compared to undetectable groups (37 versus 18%; p = 0.01) but not in Day-3 ES (17 versus 18%). Conclusion: sHLA-G in the unpooled FF does not influence significantly the implantation rate. The low percentage of detection on Day-2, even faithful for implantation, is a strong limit for a clinical application. No interest was observed for D3 sHLA-G detection. Grants: This work was supported by the European network of excellence EMBIC (contract 512040). doi:10.1016/j.jri.2007.06.027 S16 Innate immunity is linked with oxidative stress and may lead to male infertility M. Fraczek a , A. Szumala-Kakol b , P. Jedrzejczak c , M. Kurpisz a a
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; Unit of Microbiology, Poznan Hospital medical College, Poland; c Clinic of Infertility and Reproductive Endocrinology, University of Medical Sciences, Poznan, Poland
b
It was aimed to verify particular components of inflammation in order to dissect their influence on oxidative stress which directly affects spermatozoa function. In composed in vitro system, we checked the influence of selected bacterial strains (Escherichia coli, Staphylococcus haemolyticus, Streptococcus oralis, Bacteroides ureolyticus, Ureaplasma urealyticum), leukocytes and cytokines (IL-1␣, IL-6, IL-8, TNF-␣, IL-12 and IL-18) alone or in combinations on peroxidative lipid production on spermatozoal membranes. Lipid peroxidation product was evaluated through determination of the malondiladehyde (MDA) levels with high-performance lipid chromatography (HPLC) in sperm lysates. Incubation was carried out with inflammatory components, i.e. co-incubated cell suspen-
A11
sions of sperm with leukocytes, were subjected to addition of particular bacteria strains or cytokines (alone or in combinations) and its effect on lipid peroxidation was subsequently evaluated. Leukocytes were first removed from the cell suspension in order the measurable effect on sperm lipids only was assessed. Among the tested inflammatory factors, leukocytes and bacteria seemed to have the greatest influence on the MDA level (0.48 ± 0.24 and 0.45 ± 0.22 M/l). None of the cytokines alone had influence on MDA level in spermatozoa. However, incubation of spermatozoa with cytokines and leukocytes together exerted statistically significant effect in comparison to cytokines with spermatozoa only. Some combinations of cytokines (e.g., IL-6 + IL-8) influenced greater effects on spermatozoa than the others reaching a statistical significance. Among the examined bacterial strains, E. coli and B. ureolyticus had the greatest influence on MDA concentration in spermatozoa membranes (p < 0.001). Addition of leukocytes to bacteria (all strains) again increased the harmful oxidative stress effect in statistically significant fashion. In summary, the most potent detrimental factors for spermatozoa function have been leukocytes and bacteria with a direct harmful effect on spermatozoa membranes. Leukocytes turned out to be a principal modulator of the activity of the other inflammatory factors; however, the harmful effect of bacteria (on spermatozoa) does not need to be mediated through leukocytes. Cytokines, in turn, seem to be dependent on the other two factors in order to be involved in the enhancement of oxidative stress reactions. It may be hypothesized that extended (not properly treated) inflammatory status of semen is linked to deleterious (to sperm function) oxidative stress which may provoke persistent male infertility. Dissection of inflammatory factors may identify the kinetics of the observed inflammatory reaction. doi:10.1016/j.jri.2007.06.028 S17 Neuronal apoptosis evaluation after intramuscular betametasone in pregnant Wistar rats M. Santucci a , S. Daher a , M. Avedissian b , D.B. Pares a , C. Jaqueta b , L.E.A.M. Mello b , A.F. Moron a a Department of Obstetrics, S˜ao Paulo Federal University, S˜ao Paulo, SP, Brazil; b Department of Physiology, Division of Neurophysiology, S˜ao Paulo Federal University, S˜ao Paulo, SP, Brazil
Objective: We investigated perinatal mortality and neuronal apoptosis in cortex and hypoccampus of newborns after betametasone intramuscular injection at second third of pregnancy of rats Wistar EPM-1. Study design: These neuronal apoptosis were assayed in 98 newborns, that were treated with intramuscular betametasone in two different doses, therapeutic dose—DT (20 mg/kg) and three times therapeutic dose—3DT (60 mg/kg), and 44 newborns from five pregnant rats were placebo-treated (control subjects—CTR). The neuronal apoptosis were assessed by immunofluorescence TUNEL method (Roche® ). The encephalic area of apoptotic measure were hippocampus (CA1, CA2, CA3 and Dentate Girus—DG), amigdala and cortex. The results were expressed by mean and standard deviation and were submitted at statistical analysis by ANOVA. Results: All rat brain was analyzed, and the most important results were observed at CA1 being 13.8% in CTR; 7.1% in DT and 10.2% in 3DT group; and at DG, the results were 4.3% in CTR; 4.0 in DT and 8.0 in 3DT group (P < 0.05). There were no significant differences of neuronal apoptosis at CA2, CA3, amigdala and cortex. Conclusion: Changes in neuronal apoptosis at CA1 and DG are associated significantly with administration of intramuscular betametasone in pregnant rats. These data support the concept of a significant role of antenatal corticosteroids administration in neuronal apoptosis. Financial support: FAPESP. doi:10.1016/j.jri.2007.06.029
Introduction: We set a multicentric blind study to document if soluble (s)HLA-G detection in either individual follicular fluids (FF) or embryo culture supernatants (ES) could be a marker of any potential of clinical pregnancy. Materials and methods: One hundred and eighty-eight individual follicular fluids, 358 Day-2 embryo culture supernatants (Day-2ES) (Center 1) and 595 day-3 embryos supernatants (Day-3 ES) (Center 2) were collected, anonymised and blindly analyzed. Traceability from fully documented embryos was established up to the implantation for 142 FF, 148 Day-2 and 177 Day-3 ES using the Web-based database (Medifirst). The clinical implantation rate of the embryo corresponding to each tested sample was defined as the number of yolk sacs/number of embryos transferred. sHLA-G was blindly detected using an optimized chemioluminescent ELISA assay (Center 3). Threshold of detection was determined in each experiment by the cut-off leading to less than 1 false positive on 60 blanks. The outcomes of the study were: the sHLA-G percentages and medians of detection in FF, Day2 and Day-3 ES; the TOP (4–5 cells on Day-2, 8–9 on Day-3, less than 10% of fragmentation, regular cells) or no TOP (other patterns) embryo repartition; the individual embryo clinical implantation rates (IR) according to the sHLA-G detection in either unpooled FF or singly cultured ES. Results: sHLA-G in FF, D2 and D3 ES were detected in respectively 66% (median: 45 ng/ml), 18.7% (median: 0 ng/ml) and 44% (median: 0 ng/ml) of the samples. FF and Day-2 sHLA-G concentrations were not correlated for the same embryo. TOP and nonTOP embryo repartitions were not influenced by sHLA-G concentrations in FF, Day-2 and Day-3 ES. In the FF, sHLA-G concentrations do not significantly influence the implantation rates even if a tendency to higher implantation rate is observed above 30 ng/ml (8.6% versus 16.9%; p = 0.07). For Day-2 ES, IR was higher in the detectable group compared to undetectable groups (37 versus 18%; p = 0.01) but not in Day-3 ES (17 versus 18%). Conclusion: sHLA-G in the unpooled FF does not influence significantly the implantation rate. The low percentage of detection on Day-2, even faithful for implantation, is a strong limit for a clinical application. No interest was observed for D3 sHLA-G detection. Grants: This work was supported by the European network of excellence EMBIC (contract 512040). doi:10.1016/j.jri.2007.06.027 S16 Innate immunity is linked with oxidative stress and may lead to male infertility M. Fraczek a , A. Szumala-Kakol b , P. Jedrzejczak c , M. Kurpisz a a
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; Unit of Microbiology, Poznan Hospital medical College, Poland; c Clinic of Infertility and Reproductive Endocrinology, University of Medical Sciences, Poznan, Poland
b
It was aimed to verify particular components of inflammation in order to dissect their influence on oxidative stress which directly affects spermatozoa function. In composed in vitro system, we checked the influence of selected bacterial strains (Escherichia coli, Staphylococcus haemolyticus, Streptococcus oralis, Bacteroides ureolyticus, Ureaplasma urealyticum), leukocytes and cytokines (IL-1␣, IL-6, IL-8, TNF-␣, IL-12 and IL-18) alone or in combinations on peroxidative lipid production on spermatozoal membranes. Lipid peroxidation product was evaluated through determination of the malondiladehyde (MDA) levels with high-performance lipid chromatography (HPLC) in sperm lysates. Incubation was carried out with inflammatory components, i.e. co-incubated cell suspen-
A11
sions of sperm with leukocytes, were subjected to addition of particular bacteria strains or cytokines (alone or in combinations) and its effect on lipid peroxidation was subsequently evaluated. Leukocytes were first removed from the cell suspension in order the measurable effect on sperm lipids only was assessed. Among the tested inflammatory factors, leukocytes and bacteria seemed to have the greatest influence on the MDA level (0.48 ± 0.24 and 0.45 ± 0.22 M/l). None of the cytokines alone had influence on MDA level in spermatozoa. However, incubation of spermatozoa with cytokines and leukocytes together exerted statistically significant effect in comparison to cytokines with spermatozoa only. Some combinations of cytokines (e.g., IL-6 + IL-8) influenced greater effects on spermatozoa than the others reaching a statistical significance. Among the examined bacterial strains, E. coli and B. ureolyticus had the greatest influence on MDA concentration in spermatozoa membranes (p < 0.001). Addition of leukocytes to bacteria (all strains) again increased the harmful oxidative stress effect in statistically significant fashion. In summary, the most potent detrimental factors for spermatozoa function have been leukocytes and bacteria with a direct harmful effect on spermatozoa membranes. Leukocytes turned out to be a principal modulator of the activity of the other inflammatory factors; however, the harmful effect of bacteria (on spermatozoa) does not need to be mediated through leukocytes. Cytokines, in turn, seem to be dependent on the other two factors in order to be involved in the enhancement of oxidative stress reactions. It may be hypothesized that extended (not properly treated) inflammatory status of semen is linked to deleterious (to sperm function) oxidative stress which may provoke persistent male infertility. Dissection of inflammatory factors may identify the kinetics of the observed inflammatory reaction. doi:10.1016/j.jri.2007.06.028 S17 Neuronal apoptosis evaluation after intramuscular betametasone in pregnant Wistar rats M. Santucci a , S. Daher a , M. Avedissian b , D.B. Pares a , C. Jaqueta b , L.E.A.M. Mello b , A.F. Moron a a Department of Obstetrics, S˜ao Paulo Federal University, S˜ao Paulo, SP, Brazil; b Department of Physiology, Division of Neurophysiology, S˜ao Paulo Federal University, S˜ao Paulo, SP, Brazil
Objective: We investigated perinatal mortality and neuronal apoptosis in cortex and hypoccampus of newborns after betametasone intramuscular injection at second third of pregnancy of rats Wistar EPM-1. Study design: These neuronal apoptosis were assayed in 98 newborns, that were treated with intramuscular betametasone in two different doses, therapeutic dose—DT (20 mg/kg) and three times therapeutic dose—3DT (60 mg/kg), and 44 newborns from five pregnant rats were placebo-treated (control subjects—CTR). The neuronal apoptosis were assessed by immunofluorescence TUNEL method (Roche® ). The encephalic area of apoptotic measure were hippocampus (CA1, CA2, CA3 and Dentate Girus—DG), amigdala and cortex. The results were expressed by mean and standard deviation and were submitted at statistical analysis by ANOVA. Results: All rat brain was analyzed, and the most important results were observed at CA1 being 13.8% in CTR; 7.1% in DT and 10.2% in 3DT group; and at DG, the results were 4.3% in CTR; 4.0 in DT and 8.0 in 3DT group (P < 0.05). There were no significant differences of neuronal apoptosis at CA2, CA3, amigdala and cortex. Conclusion: Changes in neuronal apoptosis at CA1 and DG are associated significantly with administration of intramuscular betametasone in pregnant rats. These data support the concept of a significant role of antenatal corticosteroids administration in neuronal apoptosis. Financial support: FAPESP. doi:10.1016/j.jri.2007.06.029