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

Franck Pellestor1,3, Brigitte Andréo1, Françioise Arnal2, Claude Humeau2 and Jacques Demaille1

1 CNRS-UPR 1142, F-34396 Montpellier and 2 IVF Laboratory, C.H.U. Arnaud de Villeneuve, F-34033 Montpellier, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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.

Key words: aneuploidy/chromatid predivision/human oocyte/karyotype/non-disjunction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chromosomal abnormalities are known to be the major reason for pre- and post-implantation human embryo wastage. Consequently, the direct chromosomal analysis of human gametes is an important tool for the investigation of the occurrence of these abnormalities. In sperm, considerable data have been gathered following initial investigations into the human sperm–hamster oocyte fusion technique (Rudak et al., 1978Go; Martin et al., 1987Go), and subsequently from the development of molecular cytogenetics—including fluorescence in-situ hybridization (FISH) and primed in-situ (PRINS) methodologies—with human sperm (Guttenbach and Schmid, 1991Go; Robbins et al., 1993Go; Pellestor et al., 1995Go, 1996Go). The majority of the reports have agreed that 10–13% of sperm from normal males display chromosome abnormalities (Pellestor, 1991aGo; Guttenbach et al., 1997aGo).

Data on the chromosomal constitution of human oocytes are much less abundant and consistent. The development of IVF programmes has made human oocytes available, offering the opportunity to start cytogenetic investigations on in-vitro recovered oocytes. Since 1984, more than 50 cytogenetic surveys of human oocytes have been reported, but the results have shown wide variability in the incidence of abnormalities (ranging from 8 to 59%) and contradictions concerning aetiological factors such as maternal age (Pellestor, 1991bGo; Plachot, 1997Go) or ovarian stimulation (Tarín and Pellicer, 1990Go; Gras et al., 1992Go). These divergent data might be due to the relatively small sample size of analysed oocytes in most such studies, and the difficulty of obtaining suitable metaphase spreads. Indeed, the identification of human oocyte chromosomes was often approximate (Jacobs, 1992Go). This feeling was heightened by observations (Angell, 1991Go, 1995Go, 1997Go) that single chromatids, resulting from premature centromere division at meiosis I, could constitute a major class of abnormalities in human oocytes and represent the main causal mechanism for trisomy formation. Thus, in numerous reports, single chromatids might have been mis-scored as additional chromosomes, leading to a significant bias in the assessment of non-disjunction events.

In recent years, FISH has also been used to assess aneuploidy in human oocytes (Dailey et al., 1996Go; Dyban et al., 1996Go; Wall et al., 1996Go; Martini et al., 1997Go). Although this approach overcame the difficulty of chromosome spreading, the information obtained was limited to the targeted chromosomes (from 2 to 6), and the detection of chromosomal structural abnormalities was nearly impossible. Also, inefficient or artefactual hybridization cannot be excluded, and the interpretation of FISH signals on oocytes might be ambiguous because of the possibility of overlap of the fluorescent signals (Martini et al., 1997Go). The use of spectral karyotyping employing 24 chromosome-specific painting probes has been tested on human oocytes as an interesting alternative to conventional multi-FISH procedure, but preliminary results have indicated that this approach also necessitates good quality chromosome spreads and much more time for the chromosomal identification process (Marquez et al., 1998Go). In addition, the cost of spectral karyotyping remains high, and consequently this approach is not presently accessible to all laboratories (Bezrookove et al., 2000Go).

Conventional karyotyping methods remain preferable for studying the chromosome complement of human oocytes. An air-drying fixation method has been introduced which allows oocytes to be gradually fixed onto slides, thereby limiting the scattering and artefactual loss of chromosomes (Mikamo and Kamiguchi, 1983Go). This method was successfully adapted for human oocytes (Angell et al., 1993Go; Kamiguchi et al., 1993Go; Lim et al., 1995Go; Sengoku et al., 1997Go; Nakaoka et al., 1998Go), and led to more homogeneous data than in previous reports using Tarkowski's technique (Tarkowski, 1966Go). This fixation method has been used in association with an adapted R-banding technique (reverse heating Giemsa) (Pellestor et al., 1993Go) to perform a detailed cytogenetic survey on a large sample of human oocytes resulting from failures of IVF protocols. Legally and ethically, in-vitro unfertilized oocytes often constitute the sole available materials for direct chromosomal investigation. According to the nature of the unfertilized oocytes, two categories must be distinguished. Indeed, oocytes that fail to fertilize owing to spermatozoa dysfunction constitute a different population from those that fail to fertilize despite successful fertilization of sibling oocytes from the same cohort. The former group represents an unselected population of oocytes, whereas the latter group is a selected population of `unfertilizable' oocytes. It is therefore important to bear in mind that any extrapolation to the total follicular oocyte population remains hazardous. However, chromosomal data on naturally ovulated oocytes (Jagiello et al., 1976Go; Gras et al., 1992Go) or on superovulated but non-inseminated oocytes (Martin et al. 1986Go; Wramsby et al., 1987Go; Tarin et al., 1991Go) are very scarce and must also be interpreted with caution, due to the small number of metaphases analysed and the quality of preparations. Consequently, data drawn from the cytogenetic analysis of a large sample of unfertilized oocytes might provide valuable information on the mechanism of chromosomal abnormality occurrence in female gametes and the contribution of whole bivalent malsegregation and precocious univalent division in the genesis of these abnormalities.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and IVF procedure
The material for this study was constituted by oocytes that failed to fertilize and to cleave after in-vitro insemination. These oocytes were obtained over a 3-year period from 792 patients (1036 IVF cycles) participating in the IVF programme at the Montpellier University Hospital for various indications: tubal factor (34.2%); male infertility factor (31.0%); idiopathic infertility (13.6%); endometriosis (6.2%); immunological infertility (2.1%); male infertility + endometriosis (4.0%); male infertility + tubal factor (6.4%); polycystic ovarian syndrome (1.3%); and dysovulation (1.2%). The mean age of the patients at the time of oocyte recovery was 33.7 ± 4.7 (range 19–46) years. All women involved in this study were counselled about the procedures and provided consent for their oocytes to be donated for this analysis. Details of IVF procedure including follicle stimulation, oocyte recovery, fertilization and culture conditions have been described elsewhere (Audibert et al., 1991Go; Galtier-Dereure et al., 1996Go). Briefly, two types of hormonal stimulation were used: (i) clomiphene citrate and HMG; or (ii) GnRH agonist and HMG. Oocyte–cumulus complexes were obtained via laparoscopy or ultrasound-guided transvaginal follicular aspiration. The oocytes were inseminated with 5x103 to 104 motile sperm after a pre-incubation period of 3–5 h. The presence of pronuclei was assessed 18–20 h after insemination. Oocytes which did not show fertilization were maintained in culture for a further 22 h and then prepared for chromosomal analysis, if no evidence for fertilization or cleavage was observed. Before fixation, the morphology of all oocytes and the presence of polar body were assessed using phase-contrast microscopy.

Cytogenetic analysis
Each oocyte was placed in hypotonic solution (0.9% sodium citrate, 30% fetal bovine serum) at 37°C for 15 min. The oocyte was transferred first to fresh fixative I (methanol:acetic acid:distilled water; 5:1:4, v/v) for 5 min, and then to a grease-free slide and gently covered with a flow of fresh fixative II (methanol:acetic acid; 3:1, v/v). The slide was immediately immersed in a Coplin jar containing fixative II for 10 min, then transferred to a second jar containing fixative III (methanol:acetic acid:distilled water; 3:3:1, v/v) for 1 min. Finally, the slide was slowly removed from fixative III and air-dried with moist warm air. After fixation, the slide was examined by light microscopy (x10 and x40 magnification) to check for the presence of a metaphase plate. An R-banding procedure was performed on each slide with visible chromosomes, according to a previously published protocol (Pellestor et al., 1993Go) in order to facilitate chromosomal identification.

Each chromosome preparation was examined by two investigators. Several types of chromosomal abnormalities were considered:

  1. Non-disjunction of bivalent chromosome(s) with unambiguous extra or missing whole chromosome(s).
  2. Non-disjunction by precocious division of univalent(s) illustrated by the presence of single chromatid(s), in addition to or replacing whole chromosome(s).
  3. Complex aneuploidy corresponding to the presence of the two above types of abnormality on the same chromosome plate.
  4. Extreme aneuploidy with a number of chromosomes <18.
  5. Diploidy with a 46,XX karyotype or complement with a number of chromosomes within the 2n range.
  6. Polyploidy with the presence of a number of chromosomes >2n.
  7. Structural abnormalities including chromatid break (chtb), chromosome break (chrb), acentric fragment (ace) and deletion (del).
  8. Set of chromatids alone

Chromosome classification
The chromosome classification distinguishes seven chromosome groups, identified by the letters A to G on the basis of their overall length and centromere position. The group A involves chromosomes 1, 2, 3; the group B = chromosomes 4, 5; group C = chromosomes 6, 7, 8, 9, 10, 11, 12 and X; group D = chromosomes 13, 14, 15; group E = chromosomes 16, 17, 18; group F = chromosomes 19, 20; group G = chromosomes 21, 22 and Y.

Nomenclature
Recently proposed nomenclature (Rosenbusch and Schneider, 2000Go) has been adopted in order to characterize the abnormal karyotypes arising from chromatid predivision. The abbreviation `cht' has been used as recommended by the International System for Human Cytogenetic Nomenclature. (ISCN, 1995Go) and all the individual elements (whole chromosomes and single chromatids) have been indicated in the karyotype formulation.

Statistical analysis
A {chi}2-test as used to compare the frequencies of aneuploidies. A P-value < 0.05 was considered to be significant. The Z-test for independent proportion was performed to analyse the distribution of non-disjunctions in the different chromosome groups; again, a P-value < 0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Over a 3-year period, a total of 3042 unfertilized oocytes was processed for chromosomal analysis. In 53 cases (1.7%), cells were lost during the fixation procedure, while 1146 oocytes (37.7%) were excluded because of the absence of nuclear material or a marked degeneration aspect of cells. Most of these oocytes displayed poor morphological aspect at the light microscopy level, with signs of cytoplasmic perturbations (dark or coarse cytoplasm, vesiculation, intracellular necrosis, vacuolar elements). Of the 1843 (60.6%) remaining metaphase II oocytes, definitive karyotypes were obtained in 1397 (45.9%) metaphase preparations. In total, 446 plates (14.6%) could not be correctly analysed because of excessive fragmentation of chromosomes, clustering or multiple overlapping of chromosomes, and consequently were excluded from the study.

The distribution of analysed oocytes according to IVF indications and the results of the cytogenetic analysis of the 1397 metaphases are summarized in Table IGo. A total of 1088 oocytes (77.9%) showed a normal karyotype 23,X, whereas 309 (22.1%) displayed chromosome abnormalities (Figures 1 and 2GoGo). Details of observed abnormalities are given in Table IIGo.


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Table I. Distribution of both the analysed oocytes and the obtained karyotypes
 


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Figure 1. R-banded karyotype of an aneuploid metaphase II oocyte combining hypohaploidy for chromosomes 10 and 12 and the presence of a single chromatid from a chromosome 13. The karyotype is: 22,X,-10,-12,+13cht.

 


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Figure 2. R-banded karyotype of a hyperhaploid metaphase II oocyte showing an extra chromosome 22. The karyotype is: 24,X,+22.

 

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Table II. Details of chromosomal abnormalities
 
No significant difference (P > 0.05) in the incidence of chromosomal abnormalities was observed with regard to the IVF indications. In particular, the group of 419 oocytes where failure to fertilization was directly attributed to sperm dysfunction, displayed similar global incidence of abnormalities (21.5%) and rate of aneuploidies (10.0%) compared with the other groups (Table IGo). A higher rate (28.6%) of aneuploidy was found in the group of immunological infertility, but this finding was most likely linked to the small number of karyotypes (seven) in this group (Table IGo).

Aneuploidy was observed in 151 (10.8%) oocytes. These numerical abnormalities consisted of 75 (5.4%) hypohaploid oocytes and 57 (4.1%) hyperhaploid oocytes. Due to the observation of two types of aneuploidy (i.e. single chromatid or whole chromosome malsegregation), the aneuploid oocytes were classified into four groups: 27 hypohaploidies (1.9%) with loss of whole chromosome(s); 48 hypohaploidies (3.4%) with the missing chromosome(s) replaced by single chromatid(s); 22 cases of hyperhaploidy (1.6%) with extra whole chromosome(s); and 35 hyperhaploidies (2.5%) due to the presence of extra chromatid(s). The overall frequency of hypohaploidy was not significantly different from that of hyperhaploidy ({chi}2 = 1.06; P > 0.30), but in both classes aneuploidies due to single chromatids significantly exceeded whole chromosome aneuploidies: 48 versus 27 for hypohaploidy ({chi}2 = 3.40; P < 0.05) and 35 versus 22 for hyphapoloidy ({chi}2 = 1.90; P < 0.10). Twelve (0.8%) oocytes presented numerical errors combining whole chromosome aneuploidy and single chromatid malsegregation, and were categorized as complex aneuploidy cases. Seven (0.05%) cases of extreme aneuploidy were found with a number of chromosomes ranging from 13 to 18.

Seventy-five oocytes (5.4%) displayed a diploid number of chromosomes (from 42 to 46); 64 of these oocytes clearly showed no polar body before the fixation procedure. Four cases remained ambiguous because of the presence of cellular fragments in the perivitelline space. Seven oocytes with polar body displayed unusual large size, evoking giant diploid oocytes. Four oocytes of this type were previously observed under phase-contrast microscopy (x400 magnification), after 2 min treatment in acid Tyrode solution and gently compressed between a coverslip and a prepared slide (with four small blobs of Vaseline). In one case, two distinct spindles were observed in the oocyte cytoplasm (Figure 3Go).



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Figure 3. Microscopic observation (original magnification x400) of two spindles (arrows) in the cytoplasm of a giant in-vitro unfertilized human oocyte.

 
One tetraploid metaphase was also scored. In 53 oocytes (3.8%), only sets of single chromatids (from 20 to 23) were observed. All these sets were associated with the presence of prematurely chromosome condensation (PCC) and showed evidence for the extrusion of the two polar bodies. Evident structural abnormalities were found in 29 oocytes (2.1%) and involved chromosome and chromatid breaks, deletions and acentric fragments. In six cases (0.4%), the structural abnormalities were associated with either aneuploidy or diploidy.

The distribution of non-disjunctions among chromosome groups is presented is Table IIIGo. Extreme aneuploidies were not included in this analysis since the possibility of an artefactual origin of these chromosome sets could not be totally excluded. Single chromatid and whole chromosome malsegregations have been distinguished. Among the 144 chromosome sets in which non-disjunction events were identified, the number of aneuploid chromosomes (or chromatids) ranged from one to three. Consequently, the total number of segregation errors indexed in Table IIIGo was 182. In each group, observed frequencies of aneuploidies were compared with the estimated frequencies and the expected frequencies of aneuploidy for an equal distribution of non-disjunctions among all chromosomes. All chromosome groups displayed aneuploidies, exhibiting both hypohaploidies and hyperhaploidies, but in the A group, no abnormality of single chromatids was observed. According to the chromosome groups, ratios between hypohaploidies and hyperhaploidies were variable with a slight excess of hypohaploidy in the A, B, E and G groups (ratios of 1:4, 1:5, 9:11 and 11:15 respectively). Observed and estimated frequencies of aneuploidy did not display a significant difference (P > 0.05). When compared with the theoretical expected values, no significant differences (P > 0.05) were observed in either the C, D or F groups. The A and B groups both displayed a significantly lower incidence of aneuploidy than expected, whereas the E and G groups exhibited significantly higher frequencies than expected. Details of statistics are included in Table IIIGo.


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Table III. Distribution of non-disjunctions among the seven groups of human chromosomes in aneuploid oocyte chromosome sets
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study constitutes the most extensive cytogenetic study performed on in-vitro unfertilized human oocytes to date. Despite the high number of processed cells, the rate of successfully karyotyped oocytes remains low (45.9%), because of the variable quality of available oocytes and the use of strict criteria for selection of interpretable metaphases. The source of the oocytes under study, i.e. failure of IVF, is without a doubt an important parameter implicated in the poor quality of a large part of the recovered oocytes. It is also important to distinguish between an unselected population of oocytes that fail to fertilize following insemination with dysfunctional spermatozoa and a selected population of unfertilizable oocytes obtained from a cohort in which sibling oocytes are fertilized, because this difference in origin could affect the result of the chromosomal analysis. Technical factors such as the duration of the in-vitro oocyte culture before spreading, or the superovulation treatment, have been suspected to affect the rate of chromosomal abnormalities. The principal effect of in-vitro ageing is spindle instability and chromosome scattering, but some investigations have proven the integrity of oocytes cultured in vitro for up to 72 h (Ortiz et al., 1982Go; Gifford et al., 1987Go; Payne et al., 1997Go). With regard to the effect of follicular stimulation, a large body of published evidence has failed to demonstrate any correlation between the various stimulation regimens and chromosomal abnormalities in resulting oocytes (Pieters et al., 1991Go; Plachot, 1997Go). However, the complete innocuousness of IVF processes cannot be established with certainty because of the multiplicity of technical parameters. It is therefore important to emphasize that the present study used less than optimal material, and this should be remembered when interpreting the reported data.

The success of cytogenetic analysis of human oocytes is directly dependent on the chromosome preparation. In the past, the Tarkowski technique (Tarkowski, 1966Go) was used for chromosomal preparation (Pellestor and Sele, 1988Go; Pellestor, 1991bGo), but in the present study preparations were performed exclusively with the gradual fixation method (Mikamo and Kamiguchi, 1983Go). The present authors' personal experience with the two techniques, and comparison of the results in terms of quality of the chromosome preparations obtained, supports the idea that the gradual method is the only one to guarantee reliable and efficient chromosomal analysis because of the low risk of artefactual loss of chromosomes or separation of chromatids during the fixation procedure. Supporting this assumption is the fact that previous cytogenetic studies based on the gradual fixation technique reported similar incidences of abnormalities (Table IVGo), whereas data obtained with the Tarkowski technique (for reviews, see Pellestor, 1991bGo; Kamiguchi et al., 1993Go) displayed a wide variability in the incidences of chromosomal abnormalities (from 3.0 to 66.7%) and a frequent excess of hypohaploidies. Consequently, the data obtained from most of these earlier studies might be unreliable. The same restriction could be addressed to some FISH studies performed on human oocytes which have also used the Tarkowski technique for chromosome preparation (Dailey et al., 1996Go; Benzacken et al., 1998Go; Marquez et al., 1998Go).


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Table IV. Summary of the previous cytogenetic studies of human oocytes using the gradual fixation method
 
The global incidence of chromosomal abnormalities found in the present oocyte sample was 22.1%. No significant difference in the incidence of chromosomal abnormalities was detected between the different groups of indications, except the (probably) anecdotal excess of aneuploidies in the small group of immunological infertility. A few reports have previously suggested the existence of a relationship between chromosomal abnormalities and some types of infertility, especially idiopathic and male infertility (De Sutter et al., 1991Go; Selva et al., 1991Go), but these studies might have been biased by the small number of oocytes investigated and the inaccuracy of the cytogenetic analysis. More recent studies using the gradual fixation technique have failed to show this correlation (Sengoku et al., 1997Go). In 1994, a comparative cytogenetic study in two large groups of 302 unselected in-vitro unfertilized oocytes and 293 selected unfertilizable oocytes showed there to be no significant difference in the incidence of aneuploidies between the two groups (Almeida and Bolton, 1994Go). The present results are in agreement with these data.

An alternative approach has been to analyse non-inseminated oocytes. The reported frequencies of chromosomal abnormalities in this type of oocyte appeared to be not significantly different than that in oocytes remaining unfertilized in vitro, suggesting that there is no selection at fertilization against oocytes with chromosomal abnormalities (Tarín et al., 1991Go). However, this conclusion is highly speculative and must be interpreted with caution due to the small number of non-inseminated oocytes that have been analysed.

A chromosomal abnormality rate of 22% is consistent with rates previously reported in studies using the gradual fixation method (Table IVGo). Similar results were also found in earlier studies (Veiga et al., 1987Go; Bongso et al., 1988Go; Pellestor and Sele, 1988Go; Plachot et al., 1988Go), but the essential difference lies in the type and the distribution of the reported abnormalities. Indeed, in addition to the risk of chromosomal loss linked to the fixation process, the former studies did not consider the mechanism of sister chromatid predivision evidenced in human oocyte complements (Angell, 1991Go).

Premature chromatid division is a phenomenon usually observed in chromosome instability syndromes. It has also been implicated as a cause of the high frequency of aneuploidies in malignancies (Vig, 1984Go; Mehes and Buhler, 1995Go). Analysis of somatic cell lines with premature chromatid separation showed the absence of mitotic block after the treatment with colcemid, suggesting that the basis of such chromosomal instability might be a mitotic spindle checkpoint defect (Matsuura et al., 2000Go). Many proteins associated with spindle checkpoint and anaphase progression in both mitosis and meiosis have been recently identified, including nuclear proteins called `cohesins'. These proteins exert cohesion between sister chromatids (Miyazaki and Orr-Weaver, 1994Go) and oppose the splitting force mediated by microtubules on kinetochores (Nicklas, 1997Go). The sister chromatid cohesion is exerted mainly in the centromeric area, as well as via numerous sites within the chromosome arms (Michaelis et al., 1997Go). The separation of sister chromatids during anaphase is in part due to the destruction of cohesins by the anaphase-promoting complex (APC) (Holloway et al., 1993Go). Premature degradation or lack of these proteins might be responsible for the premature sister chromatid separation, as evidenced in the yeast Schizosaccharomyces pombe, where mutants deficient for the cohesion protein rec8 are defective in sister chromatid cohesion (Molnar et al., 1995Go). In mammals, the loss of sister chromatid cohesion could also be linked to the variation in centromere DNA sequence or size. Sister chromatids of different chromosomes do not separate simultaneously but do so according to their amount of centromeric heterochromatin (Vig, 1987Go). The existence of a relationship between small alphoid DNA size and meiosis I non-disjunction has been suggested by several authors (Roizès, 1992Go; Lo et al., 1999Go; Maratou et al., 2000Go). Thus, it can be hypothesized that the premature sister chromatid separation reflects the fact that small alphoid arrays do not bind enough centromere-associated cohesion proteins to maintain durable cohesion between sister chromatids. Since female meiosis seems to predispose to a less efficient spindle checkpoint mechanism than male meiosis (LeMaire-Adkins et al., 1997Go), such aberrant chromosome behaviour could be well tolerated during female meiosis as cytologically evidenced (Hunt et al., 1995Go), whereas in male meiosis the presence of a univalent blocks the meiotic progression.

The presence of single chomatids in oocyte complements poses a problem not only of interpretation, but also of nomenclature. The problem of interpretation arises when, in a metaphase, a chromosome is replaced by two single chromatids. This may be interpreted as artefactual because of the possibility of post-segregational splitting of chromatids (Kamiguchi et al., 1993Go; Dailey et al., 1996Go), or classified as aneuploidy (Angell, 1997Go). The distinction between these two possibilities may be provided by the identification of morphological (Angell, 1997Go) or sequence (O'Keefe et al., 1997Go) differences between the two free chromatids. In the two cases observed in the present samples, it was not possible to perform such a distinction. However, the two cases were classified as aneuploidy because of the large distance between the two single chromatids. The question of nomenclature is also important because there is no consensus in the cytogenetic designation of a single chromatid (`m' for monad or `cht' for chromatid) and the description of karyotype with single chromatid abnormalities. Recently, a standardized nomenclature was proposed for these abnormalities which uses the abbreviation `cht' as recommended by ISCN (ISCN, 1995Go), and rightly `indicates the total number of individual elements (defined as whole chromosomes plus chromatids) and then exactly describes the detected abnormality' (Rosenbusch and Schneider, 2000Go). Consequently, this nomenclature was adopted in the present study.

The frequency of aneuploidies in the present samples was 10.8%, which was consistent with the results of previous studies using the gradual fixation method (Table IVGo). These homogeneous data indicate that the incidence of aneuploidies in human oocytes is lower than earlier studies have suggested (~20–25%). Such an aneuploidy rate of 10.8% is in better agreement with data from spontaneous abortions and clinically recognized conceptions (Jacobs, 1992Go). Both whole chromosome non-disjunction and separate chromatids contribute to the occurrence of aneuploidy in human oocytes. This strongly contrasts with the results of Angell (1997), who observed only single chromatid non-disjunctions and concluded that unbalanced chromatids was the unique cause of aneuploidy in female meiosis, and that other reports of only whole chromosome non-disjunctions should be reinterpreted due to a lack of convincing photographic evidence (Angell, 1997Go). Such a categorical opinion has been disproved by several reports which have provided clear illustrations of extra whole chromosomes (Kamiguchi et al., 1993Go; Lim et al., 1995Go; Nakaoka et al., 1998Go) and demonstrated simultaneous occurrence of the two types of non-disjunction in human oocytes.

As reported previously (Kamiguchi et al., 1993Go), some oocytes displayed extreme hypohaploidy with only 13 to 18 chromosomes or combined chromosomal hypohaploidy and chromatid abnormalities. Although the artefactual loss of chromosomes during the fixation procedure cannot be totally excluded, various mechanisms inherent to oogenesis have been described such as anaphase lag (Martin, 1984Go) or an alteration in the cytoskeleton (Eichenlaub-Ritter et al., 1986Go), which could explain the loss of chromosomes in female meiosis. Also, the presence of univalents at the first meiotic division appears directly to affect the segregation of other chromosomes (Hunt et al., 1995Go; Cheng et al., 1998Go). The loss of chromosomes might be a natural propensity of mammalian female meiosis.

FISH studies have provided additional data on the chromosome status of human oocytes. Two or three rounds of FISH can be performed on fixed human oocytes, and the combined analysis of the first polar body provides an internal control (Dailey et al., 1996Go; Mahmood et al., 2000Go; Martini et al., 2000Go). FISH studies have confirmed the occurrence of chromatid separations, but some FISH reports have displayed unbelievably high rates of aneuploidy (from 37.0 to 44.0%) (Dyban et al., 1996Go; Martini et al., 1997Go, 2000Go), indicating that FISH data must also be considered with caution. Notwithstanding the questions of FISH errors and the limited number of targeted chromosomes (Dailey et al., 1996Go; Warburton, 1997Go), the high reported frequencies of aneuploidies may also be attributable to the simple fact that the FISH approach allows results to be obtained even from poor quality chromosome spreads that are usually discarded in karyotyping studies.

Such chromosome sets might preferentially result from atretic or degenerated oocytes, eventually containing partially decondensed sperm heads (Van Blerkom and Henry, 1992Go). Combined with the small sample size, this fact might lead to an overestimation of aneuploidy. Consequently, it is not believed that FISH is better suited for the analysis of human oocyte chromosomes because it cannot substitute for the examination of a whole chromosome preparation. In agreement with others (Wall et al., 1996Go), the present authors consider that FISH provides an effective method for re-analysing equivocal oocyte karyotypes.

Analysis of the distribution of individual aneuploidies among the seven chromosome groups revealed a significant excess of non-disjunction in the E and G groups, whereas groups A and B displayed a lower frequency than expected. Similar results were reported by others (Kamiguchi et al., 1993Go). To date, the previous assignment of aneuploidies by chromosome groups based on pooled data indicated a significant excess for all acrocentric chromosomes (groups D and G) (Pellestor, 1991bGo; Zenzes and Casper, 1992Go), but these data might be largely biased by the mis-scoring of single chromatids. The implication of single chromatids in non-disjunction events presents some particularities. No single chromatid was found for the group A chromosomes, but because of the large size of the present sample this cannot be attributed to an artefact. On the other hand, single chromatid abnormalities exceeded the number of whole chromosome aneuploidies in groups E and G (Table IIIGo). A lack of group A chromatid abnormalities and an excess of group E chromatid abnormalities followed by G group abnormalities has also been reported (Angell, 1997Go). This prevalence of chromatid aberrations in groups E and G might be consistent with the mechanism of lack of chromatid cohesion discussed above. To date, conventional non-disjunctions which also contributed to the elevated rate of aneuploidies in these two groups, have been associated with proximal reduced recombination (Sherman et al., 1991Go; Lamb et al., 1997Go). Consequently, it can be speculated that there is a particular feature in the conformation or in the centromeric DNA sequence of group E and G chromosomes which favours the occurrence of meiotic segregation errors. A similar tendency is also observed in human sperm where chromosomes 21, 22 (and to a lower extent chromosome 16) are more prone to non-disjunction (Egozcue et al., 1997Go; Guttenbach et al., 1997bGo; Shi and Martin, 2000Go). However, all the epidemiological surveys indicated the strong prevalence—and even the exclusivity—of a maternal origin in groups E and G aneuploidy (Nicolaidis and Petersen, 1998Go). Thus, it must be admitted that there is a specific female behaviour concerning the occurrence of meiotic segregation. The recently evidenced weakness of the female meiosis checkpoint mechanism could be an essential component of this behaviour (LeMaire-Adkins et al., 1997Go). Maternal ageing is also an essential factor in the analysis of the occurrence of aneuploidy in female gametes, though there is an evident lack of understanding of the biological mechanism of this phenomenon. Most previous cytogenetic studies have failed to confirm any relationship between maternal age and aneuploidy rate in human oocytes. Consequently, the investigation of this biological material needs to be optimized. Because of the importance of the present oocyte sample, an accurate analysis of the maternal ageing effect is still ongoing and will be reported subsequently.

A significant proportion of oocytes displayed diploid chromosome sets (5.4%) or haploid chromatid sets (3.8%) associated with the presence of prematurely condensed sperm chromatids. The combined presence of single chromatid sets and PCC has been reported previously (Angell et al., 1993Go; Schmiady and Kentenich, 1993Go; Lim et al., 1995Go) in similar proportions. The incidence of diploid oocytes was also within the range of published values (Table IVGo). The lack of first polar body extrusion appears to be the principal causal mechanism of diploid oocyte occurrence. With an estimated incidence of 0.06–0.2% (Gougeon, 1981Go; Munné et al., 1994Go), giant diploid oocytes remain rare events in human female gametes. Both endoreduplication in primary oocytes and cytoplasmic fusion of two oogonia may lead to the formation of mononuclear or binuclear giant metaphase II oocytes respectively. The observation of two spindles in the cytoplasm of a large oocyte (Figure 3Go) confirms the occurrence of a giant oocyte according to the postulated mechanisms. The non-extrusion of a polar body in such giant oocytes might explain the finding of a tetraploid metaphase. Both diploidy and PCC occurrence have been associated with cytoplasmic immaturity (Calafell et al., 1991Go; Almeida and Bolton, 1993Go). Experiments on mouse oocytes have highlighted the importance of synchrony between nuclear and cytoplasmic maturation to ensure the correct order of events through fertilization. In particular, a disturbance in the synthesis of proteins involved in spindle formation and cytokinesis may be responsible for the production of diploid metaphase II oocytes (Soewarto et al., 1995Go). In human oocytes, defects in the completion of maturation can be due to various parameters, whether physiological, hormonal, genetic or environmental (Tejada et al., 1992Go; Asch et al., 1995Go; Roberts and O'Neill, 1995Go; Zenzes et al., 1995Go). In the particular case of in-vitro unfertilized oocytes, additional factors linked to the IVF procedure, i.e. stimulation regimens (Tarín and Pellicer, 1990Go; Pieters et al., 1991Go) and temperature fluctuation (Almeida and Bolton, 1995Go), cytoplasmic dysmorphism (Van Blerkom and Henry, 1992Go) and disturbance in microtubule organization (Asch et al., 1995Go; Kim et al., 1998Go) might also affect the maturation kinetics and the fertilizability. All these data have significant implications for the understanding and diagnosis of various forms of infertility.

The cytogenetic investigation of human oocytes has shown that numerous chromosomally altered oocytes may progress through meiosis. Both univalent and whole non-disjunctions contribute to the high incidence of aneuploidy found in human female gametes. The mechanisms leading to aneuploidy, and generally speaking to abnormal chromosome complements in female gametes, are multiple since the female meiosis process is definitively sensitive to various exogenous and endogenous factors. The debate over the role of these mechanisms is not yet closed, and the data provided by the direct chromosomal and cytological analysis of oocytes remain essential.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by the Association Franciaise Contre les Myopathies (AFM) and a European grant COPERNICUS 2 (Contract ICA2-CT-2000-10012, proposal ICA2-1999-20007).


    Notes
 
3 To whom correspondence should be addressed at: CNRS-UPR 1142, IGH, 141, rue de la Cardonille, F-34396 Montpellier cedex 5, France. E-mail: Franck.Pellestor{at}igh.cnrs.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 19, 2001; resubmitted on December 31, 2001; accepted on April 4, 2002.