In vitro culture of early bovine embryos with trophoblastic vesicles: Cleavage through the block stage, followed by pregnancy after transfer

Abstract

To evaluate the direct effect of photodynamic treatment with 5-aminolevulinic acid (ALA) on preimplantation mouse embryos in an in vitro setting.Preimplantation mouse embryos were incubated with or without ALA for 5 hours and followed immediately by light exposure for 0, 5, or 15 minutes. Comparison of the viability and blastocyst formation was made among different treatment groups.A conventional laboratory setting with embryo culture facilities.Female CD1 mice were superovulated with pregnant mare serum gonadotropin and hCG before mating. Four- and eight-cell embryos and compacted morulae were flushed from the oviducts and incubated with 0, 0.1, 0.5, 1.0, or 5.0 mM ALA for 5 hours. Embryos subsequently were exposed to photoactivating light for 0, 5, or 15 minutes.Microscopic assessment of embryo quality at 12 hours and determination of the percentage of embryos progressing to the blastocyst stage at 36 or 60 hours.Incubation of embryos with 0.1, 0.5, 1.0, 5.0 mM ALA without light resulted in 87.3% ± 1.6%, 84.9% ± 3.4%, 81.4% ± 1.8%, and 82.8% ± 4.7% of the embryos developing to blastocysts, respectively. In the absence of ALA, light exposure for 0, 5, or 15 minutes resulted in 93.8% ± 2.3%, 92.3% ± 2.2%, and 85.9% ± 1.7% blastocyst formation. Combining treatment of ALA at the same concentrations with light resulted in 33.3% ± 2.1%, 0.7% ± 0.9%, 0%, 0% (5-minute light), 13.3% ± 1.0%, 0%, 1.6% ± 1.3%, 0% (15-minute light) blastocyst formation, respectively. When gross morphology was used to assess embryo viability at 12 hours, similar results were observed. Measurement of the fluorescent spectrum of embryos incubated with ALA indicated that protoporphyrin IX had been formed.Photodynamic ablation of mouse embryos was achieved with ALA under in vitro conditions. These results indicate that preimplantation mouse embryos are capable of converting ALA to the photosensitizer, protoporphyrin IX, and are susceptible to subsequent photoablation. A photodynamic effect on the embryo may be important to the successful application of this technique to the treatment of human ectopic pregnancy.