Konstantinos A Economou1, Dimitra Christopikou2, Erika Tsorva2, Stephen Davies2, Minas Mastrominas2, Haris Cazlaris2, Michael Koutsilieris3, Panagoula Angelogianni3, Dimitris Loutradis4. 1. Embryogenesis, Assisted Reproduction Unit, 49 Kifissias Avenue and Ziridi Street, 151 23 Maroussi, Athens, Greece. keconomou@gmail.com. 2. Embryogenesis, Assisted Reproduction Unit, 49 Kifissias Avenue and Ziridi Street, 151 23 Maroussi, Athens, Greece. 3. Department of Physiology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece. 4. First Department of Obstetrics and Gynaecology, School of Medicine, Alexandra University Hospital, National and Kapodistrian University of Athens, Athens, Greece.
Abstract
PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.
PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant humanGM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.