Inseminated oocytes must be dissected the day following insemination in order to assess fertilization. Oocytes at this time are normally covered with a layer of coronal and cumulus cells. These are carefully dissected away in order to clearly visualize the cell cytoplasm and examine for the presence of two pronuclei indicating normal fertilization. Oocytes that have had ICSI treatment have all cells removed prior to injection, and these can be directly scored for fertilization without further treatment. Scoring for pronuclei should be done between 17 and 20 hours after insemination, before pronuclei merge during syngamy (Figure 5.5) (see Chapter 6; p. 91).
Normally fertilized eggs should have two pronuclei, two polar bodies, regular shape with intact zona pellucida, and a clear healthy cytoplasm. However, a variety of different features may be observed: the cytoplasm of normally fertilized eggs is usually slightly granular, whereas the cytoplasm of unfertilized eggs tends to be completely clear and featureless. The cytoplasm can vary from slightly granular and healthy-looking, to brown or dark and degenerate. The shape of the egg may also vary, from perfectly spherical to irregular.
Single pronucleate zygotes obtained after conventional IVF have recently been analysed by fluorescence in situ hybridization (FISH) to determine their ploidy: of 16 zygotes, 10 were haploid and 6 were diploid (4 XY and 2 XX). It seems that during the course of their interaction, it is possible for human gamete nuclei to associate together and form diploid, single pronucleate zygotes. These findings confirm a newly recognized variation of human pronuclear interaction during syngamy, and the authors suggest that single pronucleate zygotes that develop after IVF with normal cleavage may be safely replaced (Levron et al., 1995).
Zygotes with normal fertilization at the time of scoring are removed from the insemination drops or wells, transferred into new dishes or plates containing pre-equilibrated culture medium, and returned to the incubator for a further 24 hours of culture. Those with abnormal fertilization such as multipronucleate zygotes must be cultured separately, so that there is no possibility of their being selected for embryo transfer. After cleavage, multipronucleate embryos are indistinguishable from normally fertilized oocytes. Although the presence of two pronuclei confirms fertilization, their absence does not necessarily indicate fertilization failure, and may instead represent either parthenogenetic activation, or a delay in timing of one or more of the events involved in fertilization. A study has shown that in 40% of oocytes with
no sign of fertilization 17-27 hours after insemination, 41% had the appearance of morphologically normal embryos on the following day, with morphology and cleavage rate similar to that of eggs with obvious pronuclei on day 1. However, 30% of these zygotes arrested on day 2, compared with only 7% of 'normally' fertilized oocytes (Plachot et al., 1987), and they showed a reduced implantation rate of 6% compared with 11.1% for zygotes that were fertilized within the normal time frame. Cytogenetic analysis of these embryos revealed a higher incidence of chromosomal anomalies (55% vs. 29%), and a high rate of haploidy (20%), confirming partheno-genetic activation. Nine per cent were triploid, and 26% mosaic (Plachot et al., 1987) (see Chapter 7; p. 110).
Delayed fertilization with the appearance of pronuclei on day 2 may also be observed, and these embryos tend to have an impaired developmental potential. Oehninger et al. (1989) suggested that delayed fertilization could be attributed to morphological or endocrine oocyte defects in 37% of their cases, and to sperm defects in 14.8%. Thirty-three per cent of the cases studied had no obvious association with either oocyte or sperm defects.
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