Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study.

RESEARCH QUESTION: Does calcium ionophore treatment of oocytes improve fertilization rate, embryo development and outcomes in specific groups of infertile couples? DESIGN: This retrospective cohort study involved 796 couples undergoing oocyte activation with calcium ionophore (A23187) after intracytoplasmic sperm injection (ICSI) between 2016 and 2018. All metaphase II oocytes were exposed to 5 µmol/l ionophore for 15 min immediately after ICSI, cultured in vitro to the blastocyst stage, and transferred to the uteri of recipients on day 5 or cryopreserved for transfer in the next cycle. The previous cycles of the same patients formed the control group. RESULTS: Among 1261 ICSI cycles and 796 ICSI-artificial oocyte activation (ICSI-AOA) cycles, implantation, positive beta-HCG, clinical pregnancy and live birth rates were significantly (P < 0.05 to P < 0.001) improved for all groups, compared with previous cycles, except live birth rate in women with primary ovarian insufficiency (POI). Compared with previous cycles, rates of blastulation (all P < 0.001) and high-quality blastocysts (P < 0.05 to P < 0.001) were increased significantly for couples with male factor (oligoasthenoteratozoospermia [OAT]), unexplained infertility and couples with both factors in the ICSI-AOA cycles. High-quality blastocyst rate was increased in couples with polycystic ovary syndrome (PCOS) (P = 0.0453). Miscarriage rates were decreased significantly (P < 0.05 to P < 0.001) in couples with OAT, PCOS and unexplained infertility in the treatment cycles. No significant differences were found for fertilization rate, embryo development or live birth rate in patients with POI between both groups. CONCLUSIONS: Artificial oocyte activation was able to 'rescue' the poor reproductive outcomes in certain types of infertile couples with history of failure to achieve pregnancy.

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic.

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis.

Morphometric assessment of in vitro matured dromedary camel oocytes determines the developmental competence after parthenogenetic activation.

The aim of the current study was to improve the selection method of camel oocytes after in vitro maturation by reducing exclusion criteria that were based only on the presence of the first polar body. A combined nuclear and morphometric assessment of camel oocytes after in vitro maturation was included to perform a judgment. The nuclear status of the oocytes, including the presence of the first polar body, meiosis I stage, and lack of nuclear materials, was investigated. The morphometric criteria that comprised the dimensions of each oocyte were as follows: diameter of the whole oocyte, including the zona pellucida (ZPO), zona pellucida thickness (ZPT), ooplasm diameter (OD), the perivitelline space (PVS) area, and PVS diameter. Among the oocytes with different nuclear status, there were no differences in ZPO and ZPT. However, oocytes with no nuclear material showed a significant reduction in OD (110.19 ± 1.4 µm) and a significant increase in PVS area (2139 ± 324.6 µm2) and PVS diameter (13.9 ± 1.96 µm) when compared with oocytes in the meiosis I stage (117.41 ± 2.85 µm, 1287.4 ± 123.4 µm2, and 8.56 ± 0.65 µm, respectively). To simplify the selection, the major difference between meiosis I and degenerated oocytes was the diameter of the PVS, which was greater than the ZPT in degenerated oocytes. Therefore, three groups were morphologically differentiated into oocytes with polar bodies (PB1), meiosis I (MI) oocytes, and degenerated oocytes. MI oocytes were able to extrude the polar body after activation but were not able to develop into blastocysts. In contrast, MI oocytes were able to develop into blastocysts after a biphasic activation protocol in which the oocytes were electrically activated and treated with ionomycin after 2 h. In conclusion, the results obtained by the morphometric assessment allowed us to develop a simple and objective classification system for in vitro matured dromedary camel oocytes, which will lead to accurate oocyte selection for the support of subsequent embryonic development.

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

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.

Synergism between phorbol myristate acetate and calcium ionophore in inducing proliferation of in vitro γ-irradiated murine lymphocytes.

The objective of this study was to analyze the in vitro effects of γ-irradiation (0-5 Gy) on lymphocyte proliferation in animals sensitive to radiation as BALB/c mice. Lymphocytes were irradiated and underwent different treatments: quiescent cells were cultured with calcium ionophore A23187 (5 min or 48 h) with or without phorbol myristate acetate (PMA); lymphocytes (control cells or incubated with A23187 and PMA) were also cultured with four mitogens that are specific to the different subpopulations to determine the degree of inhibition of the response to radiation. Results obtained indicated that in quiescent cells, A23187 and PMA treatment had a mitogenic effect, which peaked with long A23187 treatment (48 h); synergism was further demonstrated between both drugs and was enhanced with higher ionizing radiation doses. However, in both irradiated and non-irradiated mitogen-stimulated cells, A23187 (48 h) and PMA had a strong inhibitory effect on cell proliferation. In conclusion these results indicate that irradiated BALB/c mice lymphocytes respond to treatment with A23187 and PMA more actively than controls. Inhibition of the post-exposure mitogen-induced proliferative response and the synergic effect between A23187 and PMA also suggest altered PKC activation mechanisms in cell membranes. Comparing with previous studies with in vivo irradiated mice, the effects of IR in vitro were less intense.

Lysophospholipid acyltransferases and eicosanoid biosynthesis in zebrafish myeloid cells.

Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.

Analysis of clinical outcomes with respect to spermatozoan origin after artificial oocyte activation with a calcium ionophore.

PURPOSE: Fertilization failures have occurred repeatedly in reproductive centers after intracytoplasmic sperm injection (ICSI) and artificial oocyte activation (AOA) has been used to prevent it. This study was performed to investigate whether spermatozoan origin influences clinical outcomes of AOA with a calcium ionophore. METHODS: A total of 185 ICSI cycles with a history of no or low fertilization was included in this retrospective study. The outcomes of AOA after ICSI were compared with ejaculated-normal, ejaculated-oligo-astheno-terato or extracted-testicular spermatozoa. RESULTS: There were significant differences between the previous standard ICSI cycles and AOA cycles in the rate of fertilization and clinical outcomes among cases with different sperm origins. Thirty-eight healthy babies (20 singles and 18 twins, 29 cycles) were successfully delivered, and no congenital birth defects were observed. CONCLUSIONS: Most patients with a no or low fertilization history obtained an increased fertilization rate and a positive clinical outcome with AOA regardless of the origin of spermatozoa.