TIME-COURSE OF OOCYTE ACTIVATION, PRONUCLEUS FORMATION AND CLEAVAGE IN HUMAN OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

Knowledge of the timing of the stages of fertilization in humans is still limited because the time of gamete fusion is not known when pre-ovulatory or in-vitro matured cumulus-enclosed oocytes are inseminated. We therefore studied the morphological nuclear changes in 14 patients' oocytes by means of light microscopic observation at 2, 4, 6, 8, 16, 18 and at 20 h after intracytoplasmic single sperm injection (ICSI). A total of 144 metaphase II oocytes were injected with the spermatozoa of the patients' partners. Out of the 134 oocytes that survived the injection, 93 displayed two pronuclei in the course of the observation period (69%). Out of the 93 normally fertilized oocytes, 21 extruded the second polar body at 2 h after micro-injection (23%) and 63 oocytes at 4 h (68%). Pronuclei appeared as early as 6 h after ICSI in 16 normally fertilized oocytes (17%). At 8 h, 75 (80%) oocytes had two visible pronuclei, at 16 h 92 (99%), at 18 h 76 (82%) and at 20 h 63 (68%). In 24 oocytes (26%) the appearance of pronuclei was asynchronous, while the disappearance of the pronuclei was always synchronous, except in one oocyte. Nine of the 134 successfully injected oocytes showed three equal-sized pronuclei (6.7%). Four of the nine multi-pronucleated oocytes did not extrude the second polar body at all, while the time sequence of appearance of pronuclei was similar to that of the normally fertilized oocytes. From this study it can be concluded that many of the oocytes with two pronuclei complete meiosis II by 2 h and that pronucleus formation is complete in the majority of these oocytes by 8 h after ICSI.