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 platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in 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 of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority 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 after 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 period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees 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 treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates 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 not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.
Ammit
A J
AJ
Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, NSW, Australia.
O'Neill
C
C
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000143:Acids; D016022:Case-Control Studies; D003470:Culture Media; D004789:Enzyme Activation; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D016014:Linear Models; D008297:Male; D010741:Phospholipases A / Q000097:blood*; D010972:Platelet Activating Factor / Q000494:pharmacology; D011247:Pregnancy; D018873:Pregnancy Rate
D000143:Acids; D003470:Culture Media; D010972:Platelet Activating Factor; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase