Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

Pregnancy and neonatal outcomes of artificial oocyte activation in patients undergoing frozen-thawed embryo transfer: a 6-year population-based retrospective study.

PURPOSE: To evaluate the impact of artificial oocyte activation (AOA) in pregnancy and neonatal outcomes in infertile patients undergoing cryopreserved embryo transfer. METHOD: This retrospective study included 5686 patients' transferred embryos from routine intracytoplasmic sperm injection (ICSI) and 194 patients' transferred embryos from ICSI combined with AOA (ICSI-AOA) from January 2011 to December 2016. Pregnancy and neonatal outcomes of couples undergoing routine ICSI or ICSI-AOA were analyzed before and after propensity score matching. Artificial oocyte activation was performed with ionomycin. RESULTS: The pregnancy outcomes showed no significant difference in the rates of biochemical pregnancy, clinical pregnancy, implantation, miscarriage, ectopic pregnancy, multiple pregnancy, and live births between the routine ICSI and ICSI-AOA groups before and after propensity score matching, respectively. The assessment of neonatal outcomes showed no statistically significant differences in the birth defect rate, birth weight, gestational age, preterm birth rate, early-neonatal death rate, and fetal sex ratio between the two groups, and similar results were also observed in the two matched cohorts. CONCLUSION: Artificial oocyte activation with ionomycin does not adversely affect pregnancy and neonatal outcomes in patients undergoing frozen-thawed embryo transfer, which is beneficial to clinicians counseling patients on the risks of artificial oocyte activation.

Oocyte activation by calcium ionophore and congenital birth defects: a retrospective cohort study.

OBJECTIVE: To evaluate the safety of oocyte activation by calcium ionophore in cases of failed fertilization after intracytoplasmic sperm injection (ICSI) procedure with respect to birth defects. DESIGN: A retrospective cohort of pregnancies achieved by oocyte activation with calcium ionophore after ICSI (ICSI-Ca) and routine ICSI between the years 2006 and 2014. SETTING: Not applicable. PATIENT(S): The cohort included a total of 793 pregnancies: 66 (8%) were lost to follow up and 49 (6%) were ongoing pregnancies at the time of data collection. Out of the 678 available cases for analysis, 595 treatments were ICSI alone (88%) and 83 were ICSI-Ca (12%). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy and neonatal outcomes including birth defects were compared. RESULT(S): On the basis of a cohort of 595 ICSI pregnancies and 83 ICSI-Ca pregnancies, we found no difference in birth defects rate for singletons or for twins. Additionally, no significant difference was found between defect type (chromosomal aberration or structural malformations) and malformation type (heart, urogenital, and limb), between the ICSI and ICSI-Ca groups. Moreover, no significant differences were found regarding birth weight, gestational week at time of delivery, and fetal gender for singleton or twin pregnancies. CONCLUSION(S): Ca ionophore oocyte activation should be considered as a legitimate option for cases of failed or low fertilization by ICSI.

Can calcium ionophore "use" in patients with diminished ovarian reserve increase fertilization and pregnancy rates? A randomized, controlled study.

OBJECTIVE: To determine whether calcium ionophore solution can improve the fertilization rate in patients with diminished ovarian reserve whose partners have normal sperm parameters. DESIGN: Between January 2014 and August 2014, patients with diminished ovarian reserve were randomized to make artificial oocyte activation with calcium ionophore solution. SETTING: University hospital. PATIENT(S): A total of 296 patients who had diminished ovarian reserve and partners with normal sperm parameters were included in the study. INTERVENTION(S): Metaphase 2 oocytes were treated with calcium ionophore solution (GM508 Cult-Active) for 15 minutes just after intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Fertilization rate, implantation rate, clinical pregnancy rate, ongoing pregnancy rate. RESULT(S): Fertilization, implantation, pregnancy, and ongoing pregnancy rates for the calcium ionophore and control groups were 60.7% and 55.4%, 12.8% and 10.7%, 21% and 12.8%, and 10.9% and 6.1%, respectively. CONCLUSION(S): This is the first prospective, randomized, controlled study to analyze the effect of calcium ionophore solution on fertilization rate in patients with diminished ovarian reserve. We did not observe any differences in fertilization, clinical pregnancy, or ongoing pregnancy rates between the groups. We propose that fertilization ratios could not be increased by artificial oocyte activation via application of calcium ionophore solution in patients with diminished ovarian reserve. CLINICAL TRIAL REGISTRATION NUMBER: NCT02045914.

Assisted yes, but where do we draw the line?

In a recent report in Reproductive Biomedicine Online by Ebner et al., a comprehensive multi-centre study was presented on the use of a calcium ionophore, A23187, to artificially activate oocytes from patients who had poor fertilization rates in previous cycles. Under physiological conditions, the calcium increase in oocytes at activation is caused by influx and release from specific stores and ion channels, and has precise temporal, quantitative and spatial patterns. Calcium ionophores may release Ca(2+) in an uncontrolled fashion from intracellular stores that would not normally be involved in the activation process. Ionophores, including A23187, have a multitude of effects on cell homeostasis, not yet defined in oocytes, that may have long-term effects, for example on gene expression. We suspect that the successful births reported by Ebner et al. are a result of the overriding influence of the injected spermatozoa, rather than the effect of the ionophore; nevertheless, such an invasive non-physiological approach to assisted reproduction techniques is worrying, especially as epigenetic effects may result in future generations.

Health of children born through artificial oocyte activation: a pilot study.

Artificial oocyte activation (AOA) has shown to improve fertility in severe male infertility following intracytoplasmic sperm insemination (ICSI). However, the effect of AOA on the health status of children has not been studied. This pilot historical cohort study aims to evaluate physical and mental health of 79 and 89 children from 275 and 406 couples undergoing ICSI-AOA using ionomycin and conventional ICSI, respectively. The outcomes assessed were clinical pregnancy, abortion, type of delivery, and health of children (major birth defect, mental and behavior status). No significant differences were observed between the ICSI-AOA and the ICSI groups for these parameters, and the rate of major birth defects were not significantly different between the 2 groups. In this study, AOA has not imposed a greater risk on physical and mental health of children born through AOA, but for such a solid conclusion, further trails with higher number of cases are required and conclusions drawn are limited to this study.

Sperm head vacuoles are not affected by in-vitro conditions, as analysed by a system of sperm-microcapture channels.

Since the introduction of the motile sperm organelle morphology examination, there has been increasing recognition of the fact that the presence of large nuclear vacuoles might have deleterious effects on embryo development. Nevertheless, one fundamental question still being debated is whether specific in-vitro conditions during the handling of semen have an impact on vacuole formation. This study's objective was to analyse whether incubation temperature (20, 37°C) or oxidative stress stimulates the formation of nuclear vacuoles. Furthermore, it examined whether vacuoles disappear in the presence of an acrosome reaction inducer. Therefore, a system of sperm-microcapture channels was developed to permit the observation of the same living spermatozoa over a period of 24h. Neither incubation at 37°C nor induction of oxidative stress led to de-novo formation of nuclear vacuoles. Induction of the acrosome reaction using calcium ionophore A23587 did not lead to any modifications in the proportion of spermatozoa with vacuoles or to the disappearance of pre-existing vacuoles. According to these observations, it is concluded that nuclear vacuoles on the sperm head are already produced at earlier stages of sperm maturation and are not induced or modulated by routine laboratory environments. The examination of spermatozoa at very high magnification has led to the increasingly widespread recognition that the presence of large vacuoles in the human sperm head has deleterious effects on embryo development. One fundamental question, however, still remains: do specific conditions in the laboratory during the preparation and the handling of semen have an impact on vacuole formation? Our initial objective was to analyse whether different incubation temperatures (20, 37°C) and the induction of oxidative stress lead to the formation of sperm head vacuoles. Furthermore, we examined whether vacuoles disappear in the presence of an acrosome reaction inducer. In order to do this we developed a system of sperm-microcapture channels, which permits the observation of the same living spermatozoa over a period of 24h. Incubation at 37°C or induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, we did not detect any modification in the proportion of vacuolated spermatozoa. According to our observations, different temperatures or environmental conditions in the laboratory have no impact on the formation or disappearance of vacuoles. We conclude that sperm head vacuoles are already produced at earlier stages of sperm maturation.

Centrosome amplification and chromosomal instability in human and animal parthenogenetic cell lines.

Parthenotes have been proposed as a source of embryonic stem cells but they lack the centriole which is inherited through the sperm in all mammalian species, except for rodents. We investigated the centrosome of parthenotes and parthenogenetic embryonic stem cells using parthenogenetic and biparental pig pre-implantation embryos, human and pig parthenogenetic and biparental embryonic stem cells, sheep fibroblasts derived from post implantation parthenogenetic and biparental embryos developed in vivo. We also determined the level of aneuploidy in parthenogenetic cells. Oocytes of all species were activated using ionomycin and 6-dimethylaminopurine (6-DMAP). Over 60% of parthenogenetic blastomeres were affected by an excessive number of centrioles. Centrosome amplification, was observed by microscopical and ultrastructural analysis also in parthenogenetic cell lines of all three species. Over expression of PLK2 and down regulation of CCNF, respectively involved in the stimulation and inhibition of centrosome duplication, were present in all species. We also detected down regulation of spindle assembly checkpoint components such as BUB1, CENPE and MAD2. Centrosome amplification was accompanied by multipolar mitotic spindles and all cell lines were affected by a high rate of aneuploidy. These observations indicate a link between centrosome amplification and the high incidence of aneuploidy and suggest that parthenogenetic stem cells may be a useful model to investigate how aneuploidy can be compatible with cell proliferation and differentiation.

Anti-allergic activity of crystallinity controlled N-acetyl glucosamine.

CONTEXT: Chitin is the polysaccharide and is found in insects, parasites and fungi. Chitin has shown various immunological effects in in vivo and in vitro models. In this study, crystallinity controlled N-acetyl glucosamine (CCG) which has a high solubility was prepared from the low molecular weight chitosan. However, the effect of CCG in an allergic response is not clear. OBJECTIVE: To investigate the effect and regulatory mechanism of CCG on allergic responses. METHODS: To demonstrate the effect of CCG, we induced systemic anaphylactic shock, ear swelling response, passive cutaneous anaphylaxis (PCA), and inflammatory reaction. RESULTS: CCG inhibited the compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated PCA was inhibited by the oral administration or topical application of CCG. Histamine and ß-hexosaminidase release from mast cells was decreased with the treatment of CCG. CCG also inhibited phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced interleukin-1ß production and mRNA expression by suppressing NF-κB activation and IκBα phosphorylation. Furthermore, CCG suppressed the activation of caspase-1. CONCLUSION: These results suggest that CCG may have beneficial applicability as a candidate for an anti-allergic agent.