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 less plat elet-activating factor (PAF) than is optimal for development, It was p reviously shown that supplementation of culture media,vith PAF results in a significant increase in pregnancy rate, Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:ac etylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inact ive form, lyso-PAF, Thus, effective supplementation of media with PAF requires inactivation of this enzyme, Tn this study we examine the eff icacy of the methods of PAF:AH deactivation used for PAF supplementati on of IVF culture medium, When the effectiveness of a commonly used ac id treatment protocol (pH 3.0 at room temperature for 5 min) was exami ned, it was found that it was not completely effective for the majorit y of sera, When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining afte r 24 h (showing that the acid treatment was effective), 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 to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The peri od of acidification and the incubation temperature were assessed to de velop a new acid-treatment protocol (20 min acid treatment at 37 degre es C) which was able to deactivate PAF:AH effectively in all sera (53/ 53) examined, A trial was performed to assess the effect of acid treat ment of serum for 5 min at room temperature compared with the new prot ocol (20 min at 37 degrees C) on IVF outcome, following PAF supplement ation of IVF culture medium, Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately e qual numbers of embryos were transferred or cryopreserved, Pregnancy r ates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid-treatment protocol, The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does n ot have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.