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.

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.

No abstract

Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period (1986-1995) on 5032 thawed cycles involving 14 222 stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue.

Human embryos from the 2-cell to the morula stage developing in vitro after monospermic fertilization were incubated with [3H]thymidine or [3H]uridine and those possessing multinucleated blastomeres were examined by conventional transmission electron microscopy and by light- and electron-microscope autoradiography. Labelled DNA was present in all nuclei showing normal ultrastructural appearance, in pseudonuclei lacking nucleoli and their precursors and often demonstrating an unusual chromatin organization, free in the cytoplasm in structures resembling aggregates of dense chromatin and in small cytoplasmic vesicles in the close vicinity of these aggregates. The labelling with [3H]thymidine was not detected in about 50% of the cytoplasmic chromatin aggregates, suggesting that this extranuclear DNA was no longer replicated. Signs of extrusion of pseudonuclei to the extracellular space were occasionally observed. RNA synthesis could not be detected on free cytoplasmic chromatin, while [3H]uridine was incorporated into all nuclei and most pseudonuclei of multinucleated blastomeres of embryos at the 8-cell and morula stages. However, the major outburst of transcriptional activity and the ultrastructural cytoplasmic changes typical of the assumption of embryonic genome expression, occurring normally at the 8-cell stage, were observed only exceptionally.