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G-CSF pharmacologic supplementation in the ART (Assisted Reproductive Technologies) treatment cycles of low responder women
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Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
ning of 2008 with the keywords, infertility, fertility related distress, stress, in vitro fertilization. Twenty-eight studies approximately, have investigated the impact of distress on IVF outcome. Two-thirds of these studies have found a significant relationship between infertility distress and subsequent pregnancy rates. Major findings from these studies showed that the baseline and procedural levels of anxiety (acute and chronic) and depression, mood state and certain coping modes were significantly associated to numerous biological end-points, including number of oocytes retrieved and fertilized and pregnancy rate. The remaining studies either noticed a trend or did not find a relationship between distress and pregnancy outcome. There are many studies that support that psychological stress may impact on IVF outcome and specifically decreases the pregnancy rates. It is possible that treatments could combine hormone and psychological interventions. doi:10.1016/j.jri.2009.06.220 P15 Cellular immunity in the outcome of in vitro fertilization–embryo transfer V. Kitsiou b , S. Dendrinos b , K. Psarra a , V. Kapsimali a , G. Creatsas b , C. Papasteriades a a
Immunology and Histocompatibility Department, “Evangelismos” Hospital, Athens, Greece b B’ Obstetrics and Gynecology Department, University of Athens, Athens, Greece The close relationship of reproductive outcome and immune system has been recognized many years ago and has been extensively studied. In vitro fertilization (IVF) and embryo transfer (ET) are well-established techniques overcoming the problem of infertility. The rate of IVF/ET success remains low, with an elusive etiology in most cases. The aim of this study was to evaluate the involvement of cellular immunity in women with multiple failures of IVF/ET attempts, through the assessment of peripheral blood (PB) leukocyte subpopulations. The study included 55 infertile women with failed IVF treatments and 45 fertile women used as a control group. The immunophenotyping of the PB leukocyte subpopulations (CD2+, CD3+, CD3+CD4+, CD3+CD8+, CD5+CD19+, CD3−CD16/56+, CD3+TCR␥␦+, CD3−CD16+, CD3−CD56+, CD3−CD16+CD56+, CD3−CD16−CD56+) was performed in whole blood by flow cytometry. The study showed (A) a statistically significant increase CD3+TCR␥␦+ (p = 0.018), CD3−CD56+ (p = 0.005) and CD3−CD16+CD56+
(p < 0.001) cells in the study group in comparison to the control group, (B) no statistically significant difference in the CD2+, CD3+, CD3+CD4+, CD3+ CD8+, CD5+CD19+, CD3−CD16/56+, CD3−CD16+, CD3−CD16−CD56+ subpopulations between the two groups studied. In conclusion, the increase of PB CD3+ TCR␥␦+, CD3−CD56+ and CD3−CD16+CD56+ cells, known to participate in the pathogenesis of recurrent spontaneous abortions as well, may affect negatively the outcome of IVF/ET. Therefore the study of these parameters seems to contribute substantially to the pre IVF/ET immunological evaluation and may further help in manipulations of these cases. doi:10.1016/j.jri.2009.06.221 P16 G-CSF pharmacologic supplementation in the ART (Assisted Reproductive Technologies) treatment cycles of low responder women F. Scarpellini a , M. Sbracia a , A. Patella b a
Hungaria Center for Endocrinology and Reproductive Medicine (CERM), Rome, Italy b University of Ferrara, Department of Ob/Gyn, Ferrara, Italy Granulocyte-Colony Stimulating Factor (G-CSF) is a cytokine discovered as a growth factor for granulocytes but it has been shown to have a lot of other physiologic actions. Recently, its role in the reproductive systems has been pointed out and particularly in ovarian follicles where the G-CSF concentration was found to be directly correlated to the oocyte quality and this was in relationship to the patient age. In all the techniques of ART (Assisted Reproductive Technology) oocyte number recruitment is crucial and a good quality of them is mandatory for the success of the event. The most important problem for every ART procedure is the low responder patients. In order to improve the response of the ovary to the pharmacologic stimulatory treatment we administered a supplementation of G-CSF to low responder women in ART cycles. A randomized controlled study was conducted in ART undergoing women referred to II level center for Reproductive Medicine between January 2000 till to June 2008 using G-CSF or placebo. The inclusion criteria were: woman’s age ranging between 35 and 43 years, patients who had got at least two low responder cycles with the same different protocols, and FSH ≥ 15 UI/L and they had to be negative for abnormal karyotype, congenital and acquired Mulleran defects, infections, endocrine problems, autoimmune
Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
defects including antiphospholipid antibodies. Thirtythree women with these characteristics undergoing ICSI or IVF cycle, were enrolled in the study, they were randomly treated with G-CSF (Granulokine), at the subcutaneous dose of 1 mg/kg/day, starting from the cycle before the ovulation induced cycle or assigned to the control study group. Eighteen were supplemented with G-CSF and fifteen were treated with the same ovulation induction treatment but without this cytokine supplementation. Primary outcome was considered the number of oocytes and the number of embryos obtained at the oocyte pick up. Statistical analysis was performed using c square test and Students’ test for multiple comparisons. In the group treated with G-CSF we collected 83 oocytes, 46 embryos were obtained and 7 pregnancies with 5 healthy babies were delivered whereas in the control group only 34 oocytes and 12 embryos were obtained 2 pregnancies achieved and only 1 baby delivered (P < 0.01). In addition, the quality of oocytes and the ability to be fertilized by them was in relationship with G-CSF intrafollicular level. Our data show that G-CSF supplementation is effective to improve the results in the ART treatment of low responder women. This cytokine seems to have a local paracrine effect on oocytes stimulating their ability to develop under pharmacological stimulation and maybe even to be fertilized. doi:10.1016/j.jri.2009.06.222 P17 Preimplantation Factor (PIF*): non-invasive biomarker for embryo selection in IVF E. Promponas a,f,g , T. Keramitsoglou b , S. Ramu c,e , C.W. Stamatkin c,e , K. Pantos f , T. Vaxevanoglou f , S. Koussoulakos a , J. Diakakis g , M. Varla-Leftherioti b , E.R. Barnea d,e a
Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens, Athens, Greece b Immunobiology Department, “Helena Venizelou” Hospital, Athens, Greece c CARI Reproductive Institute, Chicago, IL, USA d The Society for the Investigation of Early Pregnancy (SIEP), NJ, USA e BioIncept, LLC Cherry Hill, NJ, USA1 f “Genesis Athens” Hospital, Athens, Greece g 3rd OB/GYN Clinic “Helena Venizelou” Maternity Hospital Athens, Athens, Greece Preimplantation Factor (PIF) is an embryo-derived peptide, which is detected during viable pregnancy in human and other mammals. The aim of the present study was to
159
investigate whether PIF levels in embryo culture supernatants of women undergoing IVF is associated with the IVF outcome, and to evaluate if PIF measurements can be used as a biomarker to identify viable embryos and increase implantation/pregnancy rates. Supernatant samples from 139 single embryo cultures from 45 women undergoing IVF, were analyzed. Samples were collected on day 2 or 3 post-fertilization. To each woman, 2–3 morphologically good embryos were transferred. PIF was measured by a multiplex immunoassay in the Luminex 200 platform, using Biotin-anti-PIFmAb (Bio-PIF-MAb*) and Ovalbumin-PIF conjugated microsphere beads (N109). PIF levels in samples was derived from standards using 5-parametere logistics curve and a threshold was established by measuring background from culture media alone and mean + 2SD was used as a cutoff (18.70 ng/ml). The results revealed that PIF was positive (>18.70 ng/ml) in 58 of the 144 supernatants (40.30%). In 26 women, PIF was positive in all (17) or at least one (9) of their samples (group A). In 19 women all samples were <18.70 ng/ml (group B). Implantation was achieved in 15 women in group A (57.69%). The implantation rate (no. of gestational sacs/no. of embryos transferred) was 18.98% (15/79) with live birth rate of 86.66% (13/15). In contrast, in women of group B implantation was achieved in 9 out of the 19 women (47.36%), the implantation rate was 14.75% (9/61) and the live birth rate 44.44% (5 of these women had first trimester abortion). The above findings indicate that increased PIF levels in embryo culture supernatants correlate with successful pregnancy and suggest that PIF measurement could be used for embryo selection in order to increase the probability for successful pregnancy in the IVF setting. 1 PIF*
Proprietary.
doi:10.1016/j.jri.2009.06.223
Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
ning of 2008 with the keywords, infertility, fertility related distress, stress, in vitro fertilization. Twenty-eight studies approximately, have investigated the impact of distress on IVF outcome. Two-thirds of these studies have found a significant relationship between infertility distress and subsequent pregnancy rates. Major findings from these studies showed that the baseline and procedural levels of anxiety (acute and chronic) and depression, mood state and certain coping modes were significantly associated to numerous biological end-points, including number of oocytes retrieved and fertilized and pregnancy rate. The remaining studies either noticed a trend or did not find a relationship between distress and pregnancy outcome. There are many studies that support that psychological stress may impact on IVF outcome and specifically decreases the pregnancy rates. It is possible that treatments could combine hormone and psychological interventions. doi:10.1016/j.jri.2009.06.220 P15 Cellular immunity in the outcome of in vitro fertilization–embryo transfer V. Kitsiou b , S. Dendrinos b , K. Psarra a , V. Kapsimali a , G. Creatsas b , C. Papasteriades a a
Immunology and Histocompatibility Department, “Evangelismos” Hospital, Athens, Greece b B’ Obstetrics and Gynecology Department, University of Athens, Athens, Greece The close relationship of reproductive outcome and immune system has been recognized many years ago and has been extensively studied. In vitro fertilization (IVF) and embryo transfer (ET) are well-established techniques overcoming the problem of infertility. The rate of IVF/ET success remains low, with an elusive etiology in most cases. The aim of this study was to evaluate the involvement of cellular immunity in women with multiple failures of IVF/ET attempts, through the assessment of peripheral blood (PB) leukocyte subpopulations. The study included 55 infertile women with failed IVF treatments and 45 fertile women used as a control group. The immunophenotyping of the PB leukocyte subpopulations (CD2+, CD3+, CD3+CD4+, CD3+CD8+, CD5+CD19+, CD3−CD16/56+, CD3+TCR␥␦+, CD3−CD16+, CD3−CD56+, CD3−CD16+CD56+, CD3−CD16−CD56+) was performed in whole blood by flow cytometry. The study showed (A) a statistically significant increase CD3+TCR␥␦+ (p = 0.018), CD3−CD56+ (p = 0.005) and CD3−CD16+CD56+
(p < 0.001) cells in the study group in comparison to the control group, (B) no statistically significant difference in the CD2+, CD3+, CD3+CD4+, CD3+ CD8+, CD5+CD19+, CD3−CD16/56+, CD3−CD16+, CD3−CD16−CD56+ subpopulations between the two groups studied. In conclusion, the increase of PB CD3+ TCR␥␦+, CD3−CD56+ and CD3−CD16+CD56+ cells, known to participate in the pathogenesis of recurrent spontaneous abortions as well, may affect negatively the outcome of IVF/ET. Therefore the study of these parameters seems to contribute substantially to the pre IVF/ET immunological evaluation and may further help in manipulations of these cases. doi:10.1016/j.jri.2009.06.221 P16 G-CSF pharmacologic supplementation in the ART (Assisted Reproductive Technologies) treatment cycles of low responder women F. Scarpellini a , M. Sbracia a , A. Patella b a
Hungaria Center for Endocrinology and Reproductive Medicine (CERM), Rome, Italy b University of Ferrara, Department of Ob/Gyn, Ferrara, Italy Granulocyte-Colony Stimulating Factor (G-CSF) is a cytokine discovered as a growth factor for granulocytes but it has been shown to have a lot of other physiologic actions. Recently, its role in the reproductive systems has been pointed out and particularly in ovarian follicles where the G-CSF concentration was found to be directly correlated to the oocyte quality and this was in relationship to the patient age. In all the techniques of ART (Assisted Reproductive Technology) oocyte number recruitment is crucial and a good quality of them is mandatory for the success of the event. The most important problem for every ART procedure is the low responder patients. In order to improve the response of the ovary to the pharmacologic stimulatory treatment we administered a supplementation of G-CSF to low responder women in ART cycles. A randomized controlled study was conducted in ART undergoing women referred to II level center for Reproductive Medicine between January 2000 till to June 2008 using G-CSF or placebo. The inclusion criteria were: woman’s age ranging between 35 and 43 years, patients who had got at least two low responder cycles with the same different protocols, and FSH ≥ 15 UI/L and they had to be negative for abnormal karyotype, congenital and acquired Mulleran defects, infections, endocrine problems, autoimmune
Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
defects including antiphospholipid antibodies. Thirtythree women with these characteristics undergoing ICSI or IVF cycle, were enrolled in the study, they were randomly treated with G-CSF (Granulokine), at the subcutaneous dose of 1 mg/kg/day, starting from the cycle before the ovulation induced cycle or assigned to the control study group. Eighteen were supplemented with G-CSF and fifteen were treated with the same ovulation induction treatment but without this cytokine supplementation. Primary outcome was considered the number of oocytes and the number of embryos obtained at the oocyte pick up. Statistical analysis was performed using c square test and Students’ test for multiple comparisons. In the group treated with G-CSF we collected 83 oocytes, 46 embryos were obtained and 7 pregnancies with 5 healthy babies were delivered whereas in the control group only 34 oocytes and 12 embryos were obtained 2 pregnancies achieved and only 1 baby delivered (P < 0.01). In addition, the quality of oocytes and the ability to be fertilized by them was in relationship with G-CSF intrafollicular level. Our data show that G-CSF supplementation is effective to improve the results in the ART treatment of low responder women. This cytokine seems to have a local paracrine effect on oocytes stimulating their ability to develop under pharmacological stimulation and maybe even to be fertilized. doi:10.1016/j.jri.2009.06.222 P17 Preimplantation Factor (PIF*): non-invasive biomarker for embryo selection in IVF E. Promponas a,f,g , T. Keramitsoglou b , S. Ramu c,e , C.W. Stamatkin c,e , K. Pantos f , T. Vaxevanoglou f , S. Koussoulakos a , J. Diakakis g , M. Varla-Leftherioti b , E.R. Barnea d,e a
Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens, Athens, Greece b Immunobiology Department, “Helena Venizelou” Hospital, Athens, Greece c CARI Reproductive Institute, Chicago, IL, USA d The Society for the Investigation of Early Pregnancy (SIEP), NJ, USA e BioIncept, LLC Cherry Hill, NJ, USA1 f “Genesis Athens” Hospital, Athens, Greece g 3rd OB/GYN Clinic “Helena Venizelou” Maternity Hospital Athens, Athens, Greece Preimplantation Factor (PIF) is an embryo-derived peptide, which is detected during viable pregnancy in human and other mammals. The aim of the present study was to
159
investigate whether PIF levels in embryo culture supernatants of women undergoing IVF is associated with the IVF outcome, and to evaluate if PIF measurements can be used as a biomarker to identify viable embryos and increase implantation/pregnancy rates. Supernatant samples from 139 single embryo cultures from 45 women undergoing IVF, were analyzed. Samples were collected on day 2 or 3 post-fertilization. To each woman, 2–3 morphologically good embryos were transferred. PIF was measured by a multiplex immunoassay in the Luminex 200 platform, using Biotin-anti-PIFmAb (Bio-PIF-MAb*) and Ovalbumin-PIF conjugated microsphere beads (N109). PIF levels in samples was derived from standards using 5-parametere logistics curve and a threshold was established by measuring background from culture media alone and mean + 2SD was used as a cutoff (18.70 ng/ml). The results revealed that PIF was positive (>18.70 ng/ml) in 58 of the 144 supernatants (40.30%). In 26 women, PIF was positive in all (17) or at least one (9) of their samples (group A). In 19 women all samples were <18.70 ng/ml (group B). Implantation was achieved in 15 women in group A (57.69%). The implantation rate (no. of gestational sacs/no. of embryos transferred) was 18.98% (15/79) with live birth rate of 86.66% (13/15). In contrast, in women of group B implantation was achieved in 9 out of the 19 women (47.36%), the implantation rate was 14.75% (9/61) and the live birth rate 44.44% (5 of these women had first trimester abortion). The above findings indicate that increased PIF levels in embryo culture supernatants correlate with successful pregnancy and suggest that PIF measurement could be used for embryo selection in order to increase the probability for successful pregnancy in the IVF setting. 1 PIF*
Proprietary.
doi:10.1016/j.jri.2009.06.223