Subzonal sperm insertion with aged human oocytes from failed in-vitro fertilization attempts: fertilization results and some applications.

This study was undertaken to assess the potential of aged human oocytes from failed in-vitro fertilization attempts as a model for the study of fertilization events after subzonal sperm insertion (SUZI). Criteria of aged oocyte suitability for this purpose were (i) the absence of nuclei, (ii) the presence of a polar body, (iii) the absence of cell division or fragmentation, (iv) marked ooplasmic contraction in hyperosmotic medium, and (v) rapid ooplasmic relaxation after returning into normo-osmotic medium following the micromanipulation. Micro-injection techniques were essentially the same as for SUZI with fresh oocytes. Oocytes that fused with the micro-injected spermatozoa developed pronuclei of typical internal structure. However, the number of pronuclei was often higher than that theoretically expected if each sperm nucleus incorporated into the oocyte gave rise to a single pronucleus. Thus, the use of aged oocytes implies the need for a specific method to assess the frequency of fusion in sperm samples examined. The results suggest that the method described here can be applied in a preliminary diagnostic test before a therapeutic SUZI attempt and in studies aimed at the optimization of sperm treatment protocols to increase the fusion capacity of subzonally inserted spermatozoa.