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Activation of Human Oocytes using Calcium Ionophore after ICSI Increases Fertilization and Number of Embryos for Couples with a History of Low ICSI Fertilization Rate.

This research presented at the European Society of Human Reproduction and Embryology, Tours, France, 1999.

Adams C.A., Anderson L.S. and Wood S.H. Reproductive Sciences Center, 4150 Regents Park Row, Suite 280, La Jolla, CA 92037, USA.

Introduction: Although ICSI is usually a highly successful treatment for severe male factor infertility, some couples repeatedly experience low ICSI fertilization rates (<50%). Possible causes include failure of oocyte activation resulting from sperm and/or oocyte defects. Previous studies have shown that oocytes injected with round spermatids (Vanderzwalmen et al., 1997) or round-headed sperm (Battaglia et al., 1997; Rybouchkin et al., 1997) can be activated by the calcium ionophore A23187 and result in live births. In this study we initially evaluated the effects of A23187 on unfertilized oocytes the day after ICSI in an attempt to rescue unactivated oocytes. The effect of ionophore treatment immediately after ICSI for couples with a history of repeatedly low fertilization rates was subsequently examined. Materials and methods: Aliquots of a stock solution of 10 mM A23187 (Calbiochem, CA) in DMSO were prepared and stored at -20¡C. Ionophore treatment was performed 16-20 hours after ICSI on unfertilized oocytes from patients with a 50% or less fertilization rate. Oocytes were incubated in 10 mM A23187 for 7 minutes at 37¡C in 5% CO2. After washing through drops of culture medium, oocytes were incubated 6-20 hours and reexamined for evidence of activation or fertilization i.e. the presence of pronuclei (PN) and polar bodies (PB). Fertilized oocytes were cultured 2-5 days and scored for embryo development. The same protocol was used for oocytes treated with A23187 immediately after ICSI, and fertilization check was performed 16-20 hours post ICSI. Results: A total of 69 unfertilized oocytes from 13 patients with an average ICSI fertilization rate of 34% were treated with A23187 the day after ICSI. The results at 20 hours post ionophore treatment were as follows: 29% (20/69) had fertilized (2PN and 2PB), 33% (23/69) had fertilized and entered syngamy (0PN and 2PB), 3% (2/69) were digynic (3PN and 1PB), 10% (7/69) had activated (1 PN and 2 PB) and 25% (17 /69) failed to activate (0PN and 1 PB). Of those oocytes classified as normally fertilized, 91% (39/43) cleaved by day 3 of culture and of those 56% (22/39) reached at least the 4-cell stage by day 4 of culture (72 hours post A23187). Three patients with a history of low ICSI fertilization had a fraction of their oocytes treated with A23187 one hour after ICSI. Overall 9/25 (36%) oocytes fertilized after routine ICSI compared with 10/13 (77%) after ICSI with ionophore treatment. No difference in embryo quality was observed between ionophore treated and untreated oocytes up to the blastocyst stage. Two of the three patients became pregnant resulting in one singleton and one twin live birth. Conclusions: Assisted oocyte activation using the calcium ionophore A23187 may be useful for enhancing ICSI fertilization rates both for unexpected fertilization failure and for couples with a history of low fertilization. These preliminary studies indicate A23187 treatment immediately after ICSI does not have a detrimental effect on embryo quality. Further studies are needed to fully understand the mechanism of ionophore action and any potential risks associated with it. References: Vanderzwalmen et al (1997) Hum. Reprod., 12, 1203-13. Battaglia et al. (1997) Fertil. Steril., 68, 118-122. Rybouchkin et al (1997) Fertil. Steril., 68, 1144-1147.

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