Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection

The aim of this study was to determine why oocytes remain unfertilized or develop three pronuclei after intracytoplasmic sperm injection (ICSI). Unfertilized and abnormally fertilized oocytes were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify oocyte, sperm and polar body DNA. One-pronuclear oocytes were considered to be unfertilized. A total of 285 unfertilized oocytes were examined (104 ICSI cycles). Overall, 83% of these oocytes were not activated (still at metaphase II) while 17% had activated and formed a single (female) pronucleus. About 66% of the unfertilized, metaphase II oocytes contained a swollen sperm head, indicating that the oocyte was correctly injected but had failed to activate and complete the second meiotic division. Premature chromosome condensation of the sperm DNA was evident in 6% of these metaphase II oocytes (4% of the unfertilized oocytes). The swollen sperm head was located among the oocyte chromosomes in 5% of the metaphase II oocytes. Other causes of failed fertilization in the metaphase II oocytes were the failure of sperm head decondensation (11%) and ejection of the spermatozoon from the oocyte (23%). A similar pattern was observed in one-pronuclear oocytes (52%, swollen sperm head; 28%, intact, undecondensed sperm head; 20%, ejection of the spermatozoon), which indicates that asynchronous pronuclear development does not explain the presence of one-pronuclear oocytes. A total of 41 three-pronuclear oocytes were examined and all had a single polar body, which indicates that the retention of the second polar body leads to the formation of the third pronucleus. In conclusion, this study demonstrates that: (i) the major cause of fertilization failure after ICSI is failure of oocyte activation; (ii) ejection of the spermatozoon into the perivitelline space is not a major cause of fertilization failure; and (iii) sperm head decondensation and oocyte activation after ICSI can occur independently.