Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media

Embryos produced by in-vitro fertilization (IVF) may produce lessplatelet-activating factor (PAF) than is optimal for development. It waspreviously shown that supplementation of culture media with PAF results ina significant increase in pregnancy rate. Human embryos are often culturedin media supplemented with serum containing the enzyme PAF:acetylhydrolase(PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF.Thus, effective supplementation of media with PAF requires inactivation ofthis enzyme. In this study we examine the efficacy of the methods of PAF:AHdeactivation used for PAF supplementation of IVF culture medium. When theeffectiveness of a commonly used acid treatment protocol (pH 3.0 at roomtemperature for 5 min) was examined, it was found that it was notcompletely effective for the majority of sera. When synthetic PAF was addedto 18 serum samples which had been acid treated, five had 90-100% of theoriginal PAF remaining after 24 h (showing that the acid treatment waseffective), eight had from 10-90% of the original PAF remaining after 24 h,and five samples had 0-10%. The extent to which PAF:AH was susceptible todeactivation was not associated with the activity in the serum prior totreatment, the serum oestradiol concentration, or the cause of infertility.The period of acidification and the incubation temperature were assessed todevelop a new acid-treatment protocol (20 min acid treatment at 37 degreesC) which was able to deactivate PAF:AH effectively in all sera (53/53)examined. A trial was performed to assess the effect of acid treatment ofserum for 5 min at room temperature compared with the new protocol (20 minat 37 degrees C) on IVF outcome, following PAF supplementation of IVFculture medium. Oocyte recovery, fertilization and embryo development rateswere equivalent for both groups and approximately equal numbers of embryoswere transferred or cryopreserved. Pregnancy rates were not significantlydifferent (14.6 versus 20.0%) for the two treatments, with a trend towardsa higher pregnancy rate with the new acid- treatment protocol. The resultsshow that this new procedure for acid treatment of serum in combinationwith PAF supplementation does not have detrimental effects on embryos andtheir pregnancy outcome and is therefore suitable for use in IVF.