Flaherty S P, Payne D, Swann N J, Matthews C D
Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, Australia.
Reprod Fertil Dev. 1995;7(2):197-210. doi: 10.1071/rd9950197.
The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.
受精评估是卵胞浆内单精子注射（ICSI）的重要组成部分，注射后约17小时通常使用诺马斯基微分干涉相差显微镜对卵母细胞进行检查。然而，用这种方式无法最终确定受精异常的病因，因此进行了细胞学研究以确定ICSI后受精失败和异常的原因。将未显示受精迹象、有一个原核（PN）或三个PN的卵母细胞用戊二醛固定，用Hoechst 33342染色，并通过荧光显微镜检查以识别PN、中期染色体、精子头部和极体。从170个ICSI周期中总共检查了428个未受精的卵母细胞。总体而言，这些未受精的卵母细胞中82%仍处于中期II（未激活），而其余18%已激活并具有1个PN和两个极体。大多数（71%）中期II卵母细胞含有肿胀的精子头部，这表明精子已正确注射，但卵母细胞未激活并完成第二次减数分裂。在4.3%的这些卵母细胞中，肿胀的精子头部位于中期染色体之间，而在某些情况下（6.6%），精子染色体发生了早熟染色体凝集（PCC）。这些中期卵母细胞受精失败的其他病因是精子从卵母细胞中排出（19%）和精子头部完全去浓缩失败（10%）。在1PN卵母细胞中也发现了类似的异常模式，尽管比例不同（肿胀的精子头部，51%；精子排出，19%；未去浓缩的精子头部，30%）。还检查了70个受精异常的卵母细胞，其中63个有3个PN和一个极体，表明未排出的第二极体发育成了第三极体。总之，本研究表明ICSI后受精失败的主要原因是卵母细胞激活失败，而非精子从卵母细胞中排出。同样明显的是，需要进一步研究以阐明控制注射卵母细胞中卵母细胞激活和精子头部去浓缩的机制。
