DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by onehalf at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.

This strategy enabled us to avoid 32.8% of complete fertilization failures after IVF, but not to decrease significantly the number of ICSI attempts in subsequent cycles. However, the uncertainties concerning the safety of ICSI suggest that ICSI should be used cautiously and judiciously.

A multicentric study was carried out to analyse in a large series: (i) the chromosomal status of unfertilized oocytes, (ii) errors at fertilization and (iii) the chromosomal complement of cleaved embryos. Parameters such as type of sterility, maternal age, stimulation treatment, doses of gonadotrophins administered and oocyte preincubation time before insemination were studied in relation to the incidence of chromosome abnormalities. Twenty-six per cent of the unfertilized oocytes and 29.2% of the embryos had chromosome anomalies. Maternal age significantly increased the rate of aneuploidy in oocytes: 38% in patients over 35 years (versus 24% in younger patients). Fertilization-related abnormalities were significant, i.e. 1.6% parthenogenesis and 6.4% polyploidy. Unexplained infertility was correlated with an increase in the rate of parthenogenesis (4.2%) when compared with tubal infertility (1.2%). Triploidy was found to be correlated with three parameters. A lower rate of triploidy was observed in the group of couples referred because of male sterility (1.9% versus 6.3% for tubal sterility), in HMG-treated patients (2.4% versus 7% with analogues of LHRH/HMG) and with a short 2-h preincubation time before insemination (3% versus 7.2% for greater than 2 h). A general model for natural selection against embryos carrying a chromosome imbalance was proposed.

Fertilization abnormalities (premature chromosome condensation of spermatozoa (PCC), triploidy, haploidy) were analysed in order to determine their origin. PCC occurs in 9% of unfertilized oocytes and seems to be the consequence of a failure of oocyte activation, leading to the continuing presence of cytoplasmic chromosome-condensing factors, causing the sperm nucleus to undergo chromosome condensation prematurely. This anomaly appears to be related to incomplete nuclear and/or cytoplasmic maturation. Triploid zygotes (6.5% of fertilized oocytes) display an original type of division: half of them divide into 3 and 6 cells, whereas at the same time diploid zygotes divide into 2 and then 4 cells. A cytological study, using both antitubulin antibodies and Hoechst dye, allowed us to demonstrate that they divide into 3 cells by means of a tripolar spindle. Triploidy seems to be correlated with four of 16 clinical or biological parameters examined: semen origin (fresh or frozen), type of stimulation treatment, number of oocytes recovered and embryo morphology. Haploid eggs (1.6% of inseminated oocytes) result from parthenogenetic activation. A correlation was found between a high number of recovered oocytes and triploid zygotes, and the occurrence of oocyte activation. These data show that increasing follicular recruitment decreases the overall oocyte quality and maturity leading to an overall 9% with impaired fertilization.