P–279 Effect of Ca2+ ionophore A23187 (calcymicin) on fertilization rates, embryo development and pregnancy in human assisted reproduction cycles

Study question Does Calcymicin improve reproductive outcomes of ICSI cycles in cases of fertilization failure and/or embryo blockage indications? Summary answer The application of the Calcymicin after ICSI improves reproductive outcomes, especially in cases with clinical indication of fertilization failure. What is known already According to the bibliography, deficiencies in the oocyte activation process frequently lead to failed ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) in the oocyte using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. Ca2+ Ionophores have been shown to trigger an initial Ca2+ spike in the ooplasm that activates Ca2+/Calmodulin dependent protein kinase II, which initiates the cascade of cellular events leading to oocyte activation. Previous results suggest that Ca2+ ionophore treatment can give live offspring after failed ICSI cycles. Study design, size, duration 270 oocytes collected from 17 patients who presented cycles with low fertilization rates and/or embryo blockage or poor quality embryos (according to ASEBIR’s embryo classification criteria) were retrospectively analyzed. Oocytes were divided into two groups, a control group that underwent conventional IVF/ICSI and another group that underwent an ICSI cycle with AOA. Study groups were defined according to clinical indications and subgroups according to AOA or control. All data were collected from 2017 until 2020. Participants/materials, setting, methods Among the 270 oocytes of the study sample, 142 belonged to the control group and 128 belonged to the AOA group. The AOA group oocytes were activated for 15 minutes immediately after ICSI using a prepared solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization rate and type, blastocyst formation rate, blastocyst quality, embryo kinetics, and pregnancy rates were analyzed, all of them were compared to FIV/ICSI cycles without oocyte activation (control group). Main results and the role of chance In the analyses of the whole sample of oocytes, the AOA treatment gave a fertilization rate of 72.5%, which was significantly higher compared to 53.8% of the control cycles (p = 0.002). Good quality blastocysts and pregnancy rates were also significantly higher than the control (p = 0.01). In the group with an indication of fertilization failure, a significantly higher fertilization rate was recorded compared to the control (65% and 33%, respectively). A higher rate of abnormal embryos with three pronuclei was also found compared to the control (p < 0.001). There were no significant differences in blastocyst formation rates, quality, or embryo kinetics (p > 0.05). In the group with an indication of embryo blockage/poor embryo quality, a significantly higher rate of good quality blastocysts and lower blastulation time were recorded compared to the control (p < 0.05). Limitations, reasons for caution The safety of the AOA technique with Ca2+ ionophore has not been fully demonstrated. In our study, none of the newborns had malformations, and gestational weeks and birth weights were normal. However, further studies on the safety of this technique are needed to implement it routinely in human reproduction clinics. Wider implications of the findings: According to these findings, an increase in the initial levels of calcium in the oocyte through the application of the Ca2+ ionophore A23187 after ICSI improves the results of failed assisted reproduction cycles, especially in the case of those diagnosed with fertility failure, which is a clear indication for AOA. Trial registration number Not applicable

Balanced Notch-Wnt signaling interplay is required for mouse embryo and fetal development

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics – Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

Can Utilization of Sperm DNA Fragmentation (SDA) Tests in Combination with Time Lapse Microscopy Help in Improving ICSI Implantation Rates in Unexplained Recurrent Implantation Failures Utilizing Double Stranded SDA as the Causative Factor for the same

Over 50% of intracytoplasmic sperm injection (ICSI) cycles don’t display implantation. Hence laboratories make their maximum efforts to select the best embryos as far as implantation enhancement is concerned. Further utilization of available technologies like time lapse recording have been made in a large number of artificial reproductive technology (ART) centres. Various studies that utilize embryo kinetics have implicated that time when embryo cleavage may prove to be an important factor that determines the implantation potential of an embryo. With this variety of algorithms mathematic wise have been used to forecast which the best embryos are for transfer. But the efficacy of these might be influenced by multiple confounding factors. Thus work on biomarkers that can forecast good ART warrants newer embryo selection basis. Regarding conventional ICSI, typical standard routine semen analysis involving sperm concentration, motility and morphology does not predict the implantation percentages in an ICSI cycle. Once sperm DNA fragmentation (SDA) methods were inducted they appeared to hold promise in forecasting good ART success. Although certain studies utilizing various techniques like TUNEL. SCSA, SCD proved a relation existed between DNA damage and implantation rates in ICSI but the same was contradicted by others. With this it was thought that bias between evaluation of ejaculate and motile sperm picked up for ICSI, as is known regarding absence of positive association of sperm motility and DNA fragmentation. Thus study by Casanovas et.al., tried to find if there is any correlation of single stranded (ssSDA) and double stranded (dsSDA) sperm DNA damage that might forecast ICSI success and utilizing Neutral Comet Assays along with help of time lapse technology they found that double stranded sperm influenced delay in embryo formation as seen by embryo kinetics and thus interfere with implantation rates. Reproduction of these findings might help in getting a standard for getting best embryos selected in ICSI utilizing SDA and time lapse microscopy.