In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.
This study addresses the incidence of failed (0%) and suboptimal (<50%) fertilization after intracytoplasmic sperm injection (ICSI), variation in the ICSI fertilization rate for specific couples, and the causes of fertilization failure and abnormal fertilization after ICSI. Failed fertilization occurred in only 37 of 1343 cycles (3%). The risk of failure was highest (37%) when only one oocyte was injected, and was lowest (0.8%) when five or more oocytes were collected. The incidence of suboptimal fertilization and the variation in the fertilization rate were studied in 87 couples who each had three cycles of ICSI in which four oocytes were injected with ejaculated spermatozoa. Approximately 74% of these couples achieved >50% fertilization in every cycle. Only 26% of the couples had <50% fertilization in one or more cycles, and most of these (17%) had only a single cycle with suboptimal fertilization. Only four of the 87 couples (5%) had suboptimal fertilization in all three cycles. The difference between the maximum and minimum fertilization rate for a couple was used as an index of variation of the fertilization rate. It was found that 47 couples (54%) had 0-25% variation, 33 couples (38%) had 26-50% fertilization and only seven couples (8%) had >50% variation. The causes of failed and abnormal fertilization were studied in unfertilized and abnormally fertilized oocytes after staining with Hoechst 33342. In total, 1005 unfertilized oocytes were studied, of which 828 (82%) were still at metaphase II and 177 (18%) were activated. Most of the oocytes (83%) contained a spermatozoon and, in the majority of these oocytes, the sperm head was partially or completely decondensed. Hence, failure of oocyte activation was the principal cause of fertilization failure. A similar pattern was observed in activated, unfertilized oocytes, although there was a higher incidence of intact spermatozoa in these oocytes compared with metaphase II, unfertilized oocytes. Interestingly, 56% of the activated oocytes contained a decondensed sperm head which was not processed into a male pronucleus. A total of 169 abnormally fertilized oocytes was also studied. Two anomalies were found: digyny due to retention of the second polar body and its subsequent transformation into a third pronucleus, and abnormal pronuclear size and number.
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.
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