The occurrence of aneuploidy in human: lessons from the cytogenetic studies of human oocytes.

During the last 4 decades, the cytogenetic investigation of human oocytes has never stopped to progress, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome nondisjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidies in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies, whereas most of initial studies have failed to confirm any relationship between maternal age and aneuploidy in human oocytes.

PRINS as an efficient tool for aneuploidy assessment in human oocytes and preimplantation embryos.

An ultrarapid three- and four-color primed in situ (PRINS) procedure has been developed for rapid chromosome identification and aneuploidy assessment on isolated cells. Based on the direct in situ mixing of fluorochromes (fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Cascade Blue), this multicolor PRINS procedure is described on unfertilized human oocytes and isolated human blastomeres.

Analysis of sperm aneuploidy by PRINS.

Based on the direct in situ mixing of the colors of different fluorochromes (fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Cascade Blue) incorporated in sequential primed in situ labeling (PRINS) reactions, a new multicolor PRINS procedure is described, allowing the rapid and distinct in situ labeling of three or four human chromosomes. Each PRINS reaction consists of a unique 5-min step for annealing and elongation. In combination with the 0.5 M NaOH pretreatment for simultaneous in situ denaturation and decondensation of sperm nuclei, this technique has been adapted to human sperm nuclei for the direct assessment of aneuploidy.

PNA on human sperm: a new approach for in situ aneuploidy estimation.

Peptide nucleic acids (PNAs) are a relatively new class of synthetic DNA mimics based on a peptide-like backbone. Since their introduction, PNA probes have become established as an efficient variation on the standard FISH procedure for chromosomal identification. In this report we have experimented with centromeric PNA probes on human sperm preparations. Both NaOH and DTT sperm decondensation procedures have been tested and comparative estimates of disomies X, Y and 1 have been performed in sperm from two donors using PNA, FISH and PRINS techniques. Similar results were obtained with the three methods, demonstrating the efficiency of PNA probes in the analysis of human sperm. The fast kinetics, stability and high specificity of PNA probes make PNA-based methodologies very valuable for in situ cytogenetic investigations.

Rapid chromosome detection by PRINS in human sperm.

Primed in situ (PRINS) labeling may be used for direct estimation of disomy rate in human sperm. We combined the PRINS procedure with a rapid and efficient NaOH pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. This enabled double labeling of human sperm within a 2-hr time span. Chromosome-specific primers have been defined for most human chromosomes, including those most frequently involved in aneuploidy. Using the dual-color PRINS method, we estimated incidences of disomy for 15 autosomes and the sex chromosomes in sperm samples from 6 normal fertile men. The frequency of disomy ranged from 0.28% to 0.36%. There were no significant interchromosomal differences in disomy rate, but chromosome 21 displayed a higher incidence of nondisjunction than did the other chromosomes.

Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies.

OBJECTIVE: To present and evaluate the use of a new ultra-fast multicolor primed in situ (PRINS) procedure for karyotyping human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, using combinations of specific primers for chromosomes 1, 7, 9, 16, and 18 and fluorescent nucleotides. SETTING: Sixteen unfertilized oocytes were obtained from women participating in an IVF program. PATIENT(S): Five patients undergoing an IVF-ET. INTERVENTION(S): In vitro unfertilized oocytes were fixed on slides, and sequential PRINS reactions were performed on each preparation. MAIN OUTCOME MEASURE(S): Ultrarapid in situ identification of three or four chromosomes on oocyte and polar body chromosome spreads. RESULT(S): On the basis of the direct in situ mixing of the colors of fluorochromes (FITC, TRITC, Cascade Blue) that were incorporated in sequential PRINS reactions, this method allows rapid and efficient labeling of three or four individual chromosomes. Each PRINS reaction consists of a unique 4- to 6-minute step for both in situ annealing and elongation. The procedure can be combined with fluorescence in situ hybridization (FISH) reactions. CONCLUSION(S): By simplifying the multicolor PRINS procedure, this new protocol should facilitate the use and adaptation of PRINS to chromosome screening. This approach could be used in parallel or in combination with FISH for efficient aneuploidy assessment on isolated cells.

Fluorescence in situ hybridization analysis of human oocytes: advantages of a double-labeling procedure.

OBJECTIVE: To improve the fluorescence in situ hybridization (FISH) chromosomal analysis of human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, with combinations of centromeric (or locus-specific) probes and whole-chromosome painting probes for chromosomes 9, 13, 16, 18, 21, and X. SETTING: Montpellier University Hospital. PATIENT(S): Women participating in an IVF program. INTERVENTION(S): Fifty-four in vitro unfertilized oocytes were fixed on slides, and simple or double FISH labeling procedures were performed on preparations. MAIN OUTCOME MEASURE(S): Simultaneous in situ visualization of specific domains and chromosome arms of each targeted chromosome. RESULT(S): Eight chromosomal abnormalities were identified, including two hyperhaploidies, three cases of extra single chromatid, and three cases of balanced separation of sister chromatids. Also, the double-labeling procedure allowed the avoidance of five interpretation errors, owing to additional artefactual signals. CONCLUSION(S): By ensuring precise identification of both chromosomes and single chromatids, the FISH double-labeling procedure limits the risk of erroneous interpretation and allows a more accurate cytogenetic analysis of human oocytes.

Mechanisms of non-disjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes.

BACKGROUND: Although numerous studies have been published on the chromosomal constitution of in-vitro unfertilized human oocytes, data remain highly variable and controversial because of the size of oocyte samples, technical reservations and potential misinterpretation. METHODS: A cytogenetic study was undertaken on 3042 unfertilized human oocytes recovered from 792 women participating in an IVF programme for various infertility problems. Both a gradual fixation technique and an R-banding procedure were used. RESULTS: The analysis was successful in 1397 oocytes (45.9%) for which interpretable metaphases were obtained. Of the 1397 oocyte karyotypes, 1088 (77.9%) were normal (23,X). The overall frequency of chromosomal abnormality was 22.1%. No correlation was found between the rate of abnormalities and the type of infertility. Aneuploidy was observed in 151 cells (10.8%), consisting of 5.4% hypohaploidies, 4.1% hyperhaploidies, 0.8% complex aneuploidies and 0.05% extreme aneuploidies with less than 18 chromosomes. Both whole chromosome non-disjunction and chromatid predivision contributed to the formation of aneuploid oocytes, but the numerical abnormalities due to single chromatids significantly exceeded conventional non-disjunctions. Abnormalities also included 5.4% diploid oocytes, 3.8% sets of chromatids alone and 2.1% structural aberrations. Aneuploidy was found in all chromosome groups. However, groups E and G exhibited significantly higher frequencies of non-disjunction than expected, whereas groups A and B showed a significantly low incidence of aneuploidy. CONCLUSIONS: The implication of both chromosome and chromatid abnormalities in the occurrence of non-disjunction are discussed in relation to the recent data on chromatid cohesion throughout cell division. The results were consistent with the hypothesis of an unequal occurrence of non-disjunction among the chromosome groups in female meiosis.

Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes.

The effect of maternal age on the incidence of chromosomal abnormalities was investigated on a large sample of 3,042 in vitro unfertilized human oocytes II obtained from 792 women aged 19-46 years and participating in an in vitro fertilization program for various indications. The chromosomal analysis combined a gradual fixation of oocytes and an adapted R-banding technique. A total of 1,397 interpretable karyotypes were obtained. Various types of numerical aberration were observed, involving conventional chromosome nondisjunction (3.5%), single-chromatid nondisjunction (5.9%), complex (0.8%) or extreme aneuploidy (0.5%), diploidy (5.4%), and set of single chromatids (3.8%). No significant difference was found in the mean age of women according to the various types of chromosomal abnormalities. A positive relationship was found between maternal age and the global rate of aneuploidy, in agreement with the findings of epidemiological studies. The incidence of both whole-chromosome nondisjunction and precocious chromatid separation were correlated to maternal aging but the most significant correlation was found between maternal aging and single-chromatid nondisjunction. The rate of diploidy was also correlated to a slight extent to maternal aging, whereas no correlation was found between maternal age and the rate of single-chromatid sets. These data reveal that single-chromatid malsegregation is an essential factor in the age-dependent occurrence of nondisjunction in human oocytes. Disturbance in sister-chromatid cohesion might be a causal mechanism predisposing to premature chromatid separation and subsequently to nondisjunction in female meiosis.