- Email: [email protected]
Cytokines in follicular fluids, implantation and miscarriage
Symposium Abstracts / Journal of Reproductive Immunology 90 (2011) 131–163
They provide the intercellular communication signals that govern leukocyte recruitment and function in the endometrium, and orchestrate an appropriate immune response to pregnancy. Through targeting cells in the preimplantation embryo and placenta, cytokines are also key mediators of the tissue restructuring processes essential for normal fetal growth and pregnancy success. One key cytokine regulating events in early pregnancy is GM-CSF (CSF2). On the basis of rigorous laboratory research this cytokine has now been developed for use in human reproductive medicine. Methods: Almost 20 years ago we showed expression of GM-CSF by epithelial cells lining the mouse reproductive tract and demonstrated its regulation by ovarian steroid hormones and factors in male seminal fluid introduced at coitus. Using genetic mouse models and using in vitro embryo culture and embryo transfer experiments, we defined a critical role for GM-CSF in targeting the preimplantation embryo to inhibit apoptosis and expression of stress response genes (Robertson, 2007; Chin et al., 2009), and showed that GM-CSF exposure in the periconceptual period alleviates effects of culture stress on later fetal and placental development (Sjoblom et al., 2005). In the endometrium, GM-CSF controls dendritic cells and their competence to activate the T cell response mediating immune tolerance for pregnancy (Robertson, 2007). These observations led to the question of whether GM-CSF might have clinical utility in improving embryo development and implantation in human IVF programs. Results: In preclinical studies we found that addition of recombinant GM-CSF to human embryo culture substantially improved their development to blastocyst stage (Sjoblom et al., 1999). We then formed a commercial partnership with Origio a/s (Denmark) who have recently completed a randomised multicentre clinical trial including >1300 IVF patients. The results reveal a 20% increase in embryo implantation at 12 weeks gestation, with effects most evident in women who had experienced previous miscarriage after IVF, where implantation rates were increased by 40%. On the basis of this result Origio a/s will release a first-in-class IVF embryo culture medium ‘EmbryoGen’ containing GM-CSF in July 2011. Conclusions: Our experience with GM-CSF demonstrates how conducting fundamental reproductive immunobiology research in animal models followed by human pre-clinical and clinical studies can be a fruitful path to valuable outcomes for reproductive medicine. There are important differences in embryo implantation, placental development and immunology of mouse and human pregnancy, but there are sufficient similarities – especially in the fundamental processes of immune adaptation for pregnancy – to justify use of mouse models and to expect that benefits for human infertility and subfertility will continue to emerge from this approach. Indeed it is critical that any new interventions for human reproductive medicine should have a sound scientific rationale and be thoroughly tested in animals before human use is considered. It seems likely that other cytokines and immune pathways discovered in mouse models will be the foundation for novel human therapies in the future.
133
References Chin, P.Y., Macpherson, A.M., Thompson, J.G., Lane, M., Robertson, S.A., 2009. Stress response genes are suppressed in mouse preimplantation embryos by granulocyte-macrophage colony-stimulating factor (GMCSF). Hum. Reprod. 24, 2997–3009. Robertson, S.A., 2007. GM-CSF regulation of embryo development and pregnancy. Cytokine Growth Factor Rev. 18, 287–298. Sjoblom, C., Roberts, C.T., Wikland, M., Robertson, S.A., 2005. GM-CSF alleviates adverse consequences of embryo culture on fetal growth trajectory and placental morphogenesis. Endocrinology. Sjoblom, C., Wikland, M., Robertson, S.A., 1999. Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro. Hum. Reprod. 14, 3069–3076.
doi:10.1016/j.jri.2011.06.006 S5 Cytokines in follicular fluids, implantation and miscarriage N. Lédée a,b,∗ , M. Petitbarat a,b , M. Rahmati a,b , G. a,b a,b Chaouat , S. Perrier d’Hauterive , C. Munaut a,b , J.M. Foidard a,b a b
University of Paris – Sud, France INSERM UMR 0782, University of Liège, Liège, Belgium
Introduction: Due to the predominant role of maternal factors during early embryo development, oocyte quality remains the main factor limiting the success of ART in human fertility. Only 5% of oocyte collected lead to the birth of healthy baby. Physicians are currently able to evaluate in routine the overall ovarian reserve of a patient but failed to define individual oocyte quality and competence and subsequent reliable parameters despite extensive researches. We will focus our attention on the immune environment of individual ovarian follicles, especially on granulocyte colony-stimulating factor (G-CSF, CSF3), the third member of the CSF family. Indeed, G-CSF quantified in individual follicular fluids appeared in three distinct experiments measuring simultaneously 26 cytokines/chemokines and growth factors (Ledee et al., 2008, 2010, 2011) as the noninvasive immune biomarker of oocyte competence able to predict the potentiality of subsequent birth of the corresponding embryo. Based on such result, we will present a first proof concept study aiming to detail the potential impact of FF G-CSF assessment on subsequent embryologist’s decision to either transfer, freeze or destruct corresponding embryos and the consequences on subsequent pregnancy rates. Materials and methods: A total of 563 follicular fluids (FF) have been collected individually, each oocyte fertilised and cultured singly with a traceability of the corresponding embryo. We quantified G-CSF in individual FF using a bead-based luminex-assay, corresponding to 119 fresh transferred embryos, 276 frozen embryos and 131 destructed embryos among 78 IVF/ICSI patients. The power of discrimination of FF G-CSF has been evaluated using the ROC curve methodology in multivariate logistic regression. Results: Combination of both FF G-CSF and embryo morphology (using a logistic fitting equation) appears to be more discriminant than morphology or FF G-CSF quantification alone to predict subsequent pregnancy. 35/78
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Symposium Abstracts / Journal of Reproductive Immunology 90 (2011) 131–163
patients (45%) would have had a benefit of such an approach since it would have modified the choice of the embryo to transfer allowing a potential increase of 10% for the ongoing pregnancy rates at the first fresh transfer. Moreover, only 26% of frozen embryos had a potentiality to implant and 10% of destructed embryos seemed to have the potential to implant. Using FF G-CSF combined with embryo morphology, all the cases at high risk of multiple pregnancy would have been identified in the present study. Conclusion: Based on this proof-of-concept study, the combination of FF G-CSF analysis with embryo morphology may improve the efficiency of in vitro fertilization and may have a major impact on embryo transfer policy. Prospective studies need to be performed to confirm our results.
(baseline, down regulation and oocyte retrieval), 24 h prior to a pregnancy test and at 6 weeks gestation if pregnant. Results: IL-33 and ST2 are expressed by the preimplantation embryo, first trimester placenta and decidua. ST2L expression by NK cells, T helper and cytotoxic T cells was increased as early as 24 h prior to a pregnancy test (9 days post implantation) in whose women with a successful implantation. This was not seen in those women in whom implantation did not occur. Circulating IL-33 or sST2 did not change significantly. These results are consistent with previous findings that in later pregnancy ST2L is up-regulated in normal pregnancy but not in pre-eclampsia. Conclusion: The IL-33/ST2 cytokine axis may have an important role in normal implantation and pregnancy. Keywords: IL-33; ST2; Implantation
doi:10.1016/j.jri.2011.06.007 S6 IL-33 and ST2 in implantation and pregnancy I.G. Granne ∗ , J. Southcombe, C.W.G. Redman, T. Child, I.L. Sargent Nuffield Department of Obstetrics and Gynaecology and Oxford Fertility Unit, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK Introduction: During pregnancy maternal adaptive and innate immunity is regulated to accommodate the immunologically foreign conceptus. Previously, maternal immunoregulation was perceived in terms of a bias towards adaptive T-helper (Th)2 activity with inhibition of cytotoxic Th1 responses In pregnancy-related complications such as recurrent miscarriage or pre-eclampsia, the Th2 bias associated with healthy pregnancy either fails to develop or is reversed, such that Th1 responses predominate. However, the finding that inflammation is important in early implantation events, systemic inflammation occurs in normal pregnancy and cells of the innate immune system can also be biased towards Type 1 or Type 2 cytokine production, suggests that the Th1/Th2 hypothesis is too simplistic. However a shift towards Type 2 immunity in normal pregnancy undoubtedly occurs. IL-33 is a recently described member of the IL-1 family which includes IL-1 and IL-18. Like these cytokines, IL-33 has strong immunomodulatory functions and predominantly induces the production of Type cytokines (IL-4, IL-5 and IL-13) in T cells and NK cells and increases serum immunoglobulin. Soluble ST2 is thought to act as a ‘decoy receptor’, competing with the membrane bound receptor (ST2L) for IL-33 binding. Increasing levels of sST2 may therefore negatively regulate IL-33 signalling and diminish the cytokines overall bioactivity. Methods: Expression of IL-33 and ST2 by human embryos was detected by immunofluorescence microscopy. Placental and decidual expression was detected by immunohistochemistry. In order to investigate whether changes in circulating IL-33, sST2 or membrane bound ST2L occur in early pregnancy, women undergoing an IVF cycle for male factor sub-fertility were recruited. Blood samples were taken throughout the cycle
doi:10.1016/j.jri.2011.06.008 S7 GM-CSF regulates uterine dendritic cell and macrophage maturation and antigen presentation L.M. Moldenhauer a,∗ , S.N. Keenihan a , J.D. Hayball b,c , S.A. Robertson a a
Robinson Institute, Research Centre for Reproductive Health, Faculty of Health Science, University of Adelaide, Australia b Experimental Therapeutics Laboratory, Hanson Institute, Adelaide, Australia c Sansom Institute, University of South Australia, Adelaide, Australia Introduction: Antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages are abundant populations in the uterine endometrium. DCs and macrophages are critical for the processing and presentation of paternal antigens to maternal T cells which allows development of the maternal immune tolerance required to accomodate the semi-allogeneic conceptus. Granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF), a known regulator of DC and macrophage maturation and function, is expressed by uterine epithelial cells, under regulation of steroid hormones and male seminal fluid factors. To investigate the importance of CSF2 in regulating uterine APCs, this study aimed to assess the abundance, phenotype and functionality of DCs and macrophages in Csf2-null mutant (Csf2−/−) and wild-type mice at estrus and in the periconceptual and peri-implantation periods. Methods: Csf2−/− and wild-type female mice on a C57Bl/6 (B6) background were mated to B6 males and the number, phenotype and distribution of APCs in endometrial tissue and whole uterus was evaluated at day 0.5 post-coitum (pc), day 3.5 pc and in virgin estrus mice by immunohistochemistry and flow cytometry. Macrophages were defined as F4/80+ and dendritic cells were defined as F4/80− and CD11c+ and/or CD11b+ . Phenotype was assessed by the markers MHC class II, macrosialin, scavenger receptor class I (MSR1), sialoadhesion, CD80 and CD86. The functional competence of uterine APCs to activate maternal T cells was investigated by measuring
They provide the intercellular communication signals that govern leukocyte recruitment and function in the endometrium, and orchestrate an appropriate immune response to pregnancy. Through targeting cells in the preimplantation embryo and placenta, cytokines are also key mediators of the tissue restructuring processes essential for normal fetal growth and pregnancy success. One key cytokine regulating events in early pregnancy is GM-CSF (CSF2). On the basis of rigorous laboratory research this cytokine has now been developed for use in human reproductive medicine. Methods: Almost 20 years ago we showed expression of GM-CSF by epithelial cells lining the mouse reproductive tract and demonstrated its regulation by ovarian steroid hormones and factors in male seminal fluid introduced at coitus. Using genetic mouse models and using in vitro embryo culture and embryo transfer experiments, we defined a critical role for GM-CSF in targeting the preimplantation embryo to inhibit apoptosis and expression of stress response genes (Robertson, 2007; Chin et al., 2009), and showed that GM-CSF exposure in the periconceptual period alleviates effects of culture stress on later fetal and placental development (Sjoblom et al., 2005). In the endometrium, GM-CSF controls dendritic cells and their competence to activate the T cell response mediating immune tolerance for pregnancy (Robertson, 2007). These observations led to the question of whether GM-CSF might have clinical utility in improving embryo development and implantation in human IVF programs. Results: In preclinical studies we found that addition of recombinant GM-CSF to human embryo culture substantially improved their development to blastocyst stage (Sjoblom et al., 1999). We then formed a commercial partnership with Origio a/s (Denmark) who have recently completed a randomised multicentre clinical trial including >1300 IVF patients. The results reveal a 20% increase in embryo implantation at 12 weeks gestation, with effects most evident in women who had experienced previous miscarriage after IVF, where implantation rates were increased by 40%. On the basis of this result Origio a/s will release a first-in-class IVF embryo culture medium ‘EmbryoGen’ containing GM-CSF in July 2011. Conclusions: Our experience with GM-CSF demonstrates how conducting fundamental reproductive immunobiology research in animal models followed by human pre-clinical and clinical studies can be a fruitful path to valuable outcomes for reproductive medicine. There are important differences in embryo implantation, placental development and immunology of mouse and human pregnancy, but there are sufficient similarities – especially in the fundamental processes of immune adaptation for pregnancy – to justify use of mouse models and to expect that benefits for human infertility and subfertility will continue to emerge from this approach. Indeed it is critical that any new interventions for human reproductive medicine should have a sound scientific rationale and be thoroughly tested in animals before human use is considered. It seems likely that other cytokines and immune pathways discovered in mouse models will be the foundation for novel human therapies in the future.
133
References Chin, P.Y., Macpherson, A.M., Thompson, J.G., Lane, M., Robertson, S.A., 2009. Stress response genes are suppressed in mouse preimplantation embryos by granulocyte-macrophage colony-stimulating factor (GMCSF). Hum. Reprod. 24, 2997–3009. Robertson, S.A., 2007. GM-CSF regulation of embryo development and pregnancy. Cytokine Growth Factor Rev. 18, 287–298. Sjoblom, C., Roberts, C.T., Wikland, M., Robertson, S.A., 2005. GM-CSF alleviates adverse consequences of embryo culture on fetal growth trajectory and placental morphogenesis. Endocrinology. Sjoblom, C., Wikland, M., Robertson, S.A., 1999. Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro. Hum. Reprod. 14, 3069–3076.
doi:10.1016/j.jri.2011.06.006 S5 Cytokines in follicular fluids, implantation and miscarriage N. Lédée a,b,∗ , M. Petitbarat a,b , M. Rahmati a,b , G. a,b a,b Chaouat , S. Perrier d’Hauterive , C. Munaut a,b , J.M. Foidard a,b a b
University of Paris – Sud, France INSERM UMR 0782, University of Liège, Liège, Belgium
Introduction: Due to the predominant role of maternal factors during early embryo development, oocyte quality remains the main factor limiting the success of ART in human fertility. Only 5% of oocyte collected lead to the birth of healthy baby. Physicians are currently able to evaluate in routine the overall ovarian reserve of a patient but failed to define individual oocyte quality and competence and subsequent reliable parameters despite extensive researches. We will focus our attention on the immune environment of individual ovarian follicles, especially on granulocyte colony-stimulating factor (G-CSF, CSF3), the third member of the CSF family. Indeed, G-CSF quantified in individual follicular fluids appeared in three distinct experiments measuring simultaneously 26 cytokines/chemokines and growth factors (Ledee et al., 2008, 2010, 2011) as the noninvasive immune biomarker of oocyte competence able to predict the potentiality of subsequent birth of the corresponding embryo. Based on such result, we will present a first proof concept study aiming to detail the potential impact of FF G-CSF assessment on subsequent embryologist’s decision to either transfer, freeze or destruct corresponding embryos and the consequences on subsequent pregnancy rates. Materials and methods: A total of 563 follicular fluids (FF) have been collected individually, each oocyte fertilised and cultured singly with a traceability of the corresponding embryo. We quantified G-CSF in individual FF using a bead-based luminex-assay, corresponding to 119 fresh transferred embryos, 276 frozen embryos and 131 destructed embryos among 78 IVF/ICSI patients. The power of discrimination of FF G-CSF has been evaluated using the ROC curve methodology in multivariate logistic regression. Results: Combination of both FF G-CSF and embryo morphology (using a logistic fitting equation) appears to be more discriminant than morphology or FF G-CSF quantification alone to predict subsequent pregnancy. 35/78
134
Symposium Abstracts / Journal of Reproductive Immunology 90 (2011) 131–163
patients (45%) would have had a benefit of such an approach since it would have modified the choice of the embryo to transfer allowing a potential increase of 10% for the ongoing pregnancy rates at the first fresh transfer. Moreover, only 26% of frozen embryos had a potentiality to implant and 10% of destructed embryos seemed to have the potential to implant. Using FF G-CSF combined with embryo morphology, all the cases at high risk of multiple pregnancy would have been identified in the present study. Conclusion: Based on this proof-of-concept study, the combination of FF G-CSF analysis with embryo morphology may improve the efficiency of in vitro fertilization and may have a major impact on embryo transfer policy. Prospective studies need to be performed to confirm our results.
(baseline, down regulation and oocyte retrieval), 24 h prior to a pregnancy test and at 6 weeks gestation if pregnant. Results: IL-33 and ST2 are expressed by the preimplantation embryo, first trimester placenta and decidua. ST2L expression by NK cells, T helper and cytotoxic T cells was increased as early as 24 h prior to a pregnancy test (9 days post implantation) in whose women with a successful implantation. This was not seen in those women in whom implantation did not occur. Circulating IL-33 or sST2 did not change significantly. These results are consistent with previous findings that in later pregnancy ST2L is up-regulated in normal pregnancy but not in pre-eclampsia. Conclusion: The IL-33/ST2 cytokine axis may have an important role in normal implantation and pregnancy. Keywords: IL-33; ST2; Implantation
doi:10.1016/j.jri.2011.06.007 S6 IL-33 and ST2 in implantation and pregnancy I.G. Granne ∗ , J. Southcombe, C.W.G. Redman, T. Child, I.L. Sargent Nuffield Department of Obstetrics and Gynaecology and Oxford Fertility Unit, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK Introduction: During pregnancy maternal adaptive and innate immunity is regulated to accommodate the immunologically foreign conceptus. Previously, maternal immunoregulation was perceived in terms of a bias towards adaptive T-helper (Th)2 activity with inhibition of cytotoxic Th1 responses In pregnancy-related complications such as recurrent miscarriage or pre-eclampsia, the Th2 bias associated with healthy pregnancy either fails to develop or is reversed, such that Th1 responses predominate. However, the finding that inflammation is important in early implantation events, systemic inflammation occurs in normal pregnancy and cells of the innate immune system can also be biased towards Type 1 or Type 2 cytokine production, suggests that the Th1/Th2 hypothesis is too simplistic. However a shift towards Type 2 immunity in normal pregnancy undoubtedly occurs. IL-33 is a recently described member of the IL-1 family which includes IL-1 and IL-18. Like these cytokines, IL-33 has strong immunomodulatory functions and predominantly induces the production of Type cytokines (IL-4, IL-5 and IL-13) in T cells and NK cells and increases serum immunoglobulin. Soluble ST2 is thought to act as a ‘decoy receptor’, competing with the membrane bound receptor (ST2L) for IL-33 binding. Increasing levels of sST2 may therefore negatively regulate IL-33 signalling and diminish the cytokines overall bioactivity. Methods: Expression of IL-33 and ST2 by human embryos was detected by immunofluorescence microscopy. Placental and decidual expression was detected by immunohistochemistry. In order to investigate whether changes in circulating IL-33, sST2 or membrane bound ST2L occur in early pregnancy, women undergoing an IVF cycle for male factor sub-fertility were recruited. Blood samples were taken throughout the cycle
doi:10.1016/j.jri.2011.06.008 S7 GM-CSF regulates uterine dendritic cell and macrophage maturation and antigen presentation L.M. Moldenhauer a,∗ , S.N. Keenihan a , J.D. Hayball b,c , S.A. Robertson a a
Robinson Institute, Research Centre for Reproductive Health, Faculty of Health Science, University of Adelaide, Australia b Experimental Therapeutics Laboratory, Hanson Institute, Adelaide, Australia c Sansom Institute, University of South Australia, Adelaide, Australia Introduction: Antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages are abundant populations in the uterine endometrium. DCs and macrophages are critical for the processing and presentation of paternal antigens to maternal T cells which allows development of the maternal immune tolerance required to accomodate the semi-allogeneic conceptus. Granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF), a known regulator of DC and macrophage maturation and function, is expressed by uterine epithelial cells, under regulation of steroid hormones and male seminal fluid factors. To investigate the importance of CSF2 in regulating uterine APCs, this study aimed to assess the abundance, phenotype and functionality of DCs and macrophages in Csf2-null mutant (Csf2−/−) and wild-type mice at estrus and in the periconceptual and peri-implantation periods. Methods: Csf2−/− and wild-type female mice on a C57Bl/6 (B6) background were mated to B6 males and the number, phenotype and distribution of APCs in endometrial tissue and whole uterus was evaluated at day 0.5 post-coitum (pc), day 3.5 pc and in virgin estrus mice by immunohistochemistry and flow cytometry. Macrophages were defined as F4/80+ and dendritic cells were defined as F4/80− and CD11c+ and/or CD11b+ . Phenotype was assessed by the markers MHC class II, macrosialin, scavenger receptor class I (MSR1), sialoadhesion, CD80 and CD86. The functional competence of uterine APCs to activate maternal T cells was investigated by measuring