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Genetic Analysis of the Oocyte—A Review
Placenta (2003), 24, S66–S69 doi:10.1016/S0143-4004(03)00143-7
Genetic Analysis of the Oocyte—A Review M. Plachot a CHI Jean Rostand, Laboratoire de Fecondation in vitro, 141 Grande Rue, 92311 Se`vres Cedex, France Paper accepted 28 May 2003
Chromosome number abnormalities are remarkably common in human reproduction. Most are caused by chromosomal non-disjunction and premature chromatid separation in oocyte meiosis I. Pooled data from previous studies showed that one in five oocytes that failed to fertilize after in vitro insemination was abnormal when analysed by conventional cytogenetics. Preconception genetic diagnosis, carried out on the first and second polar bodies by FISH, using 5 chromosome-specific probes (13, 16, 18, 21 and 22), showed that the rate of aneuploidy is higher in women aged 35 or over (52.1 per cent). Oocyte dysmorphy seems to have little effect on the rate of aneuploidy except for giant oocytes, which are usually diploid and may cause triploidy after fertilization. Intra- and extrafollicular influences (perifollicular microvasculature, oxygenation, the presence of residues from cigarette smoke) may disturb maturation, leading to immaturity and aneuploidy. Thus, oocyte meiosis is very sensitive to endogenous and exogenous factors that may cause the production of oocytes with chromosomal abnormalities and therefore, of abnormal zygotes. Placenta (2003), 24, S66–S69 2003 Elsevier Ltd. All rights reserved.
Data on the chromosomal constitution of human oocytes were reported for the first time in 1978, and have been more extensively reported during the last two decades. Indeed, the development of IVF made oocytes that failed to fertilize available for cytogenetic studies (for review, Plachot, 2001; Pellestor et al., 2002). Moreover, the development of preconceptional cytogenetic diagnosis for women of advanced age made it possible to analyse thousands of polar bodies (PBs), providing indirect information on the corresponding oocytes (Kuliev et al., 2002).
In 1991, Angell et al. (1991) observed that 13 per cent of haploid metaphases were hyperhaploid but that none contained extra whole chromosomes. The extra components were a single chromatid or two single chromatids replacing a whole chromosome. The authors suggested that the chromatids arose from premature centromere division at meiosis I, this abnormality being a major cause of trisomy. Premature chromatid division may be due to the premature degradation or lack of cohesins. These proteins exert cohesion between sister chromatids and oppose the splitting force mediated by microtubules on kinetochores (for review, Pellestor et al., 2002).
MECHANISMS OF NON-DISJUNCTION IN MEIOSIS IN HUMAN FEMALES Meiosis is the process by which a diploid cell (containing two copies of each pair of chromosomes) produces a haploid gamete containing a single copy of each chromosome. Meiotic non-disjunction is defined as the unbalanced segregation of whole chromosomes leading to hypo or hyperhaploidy. It may occur at meiosis I (when homologous chromosomes segregate to opposite poles) or at meiosis II (when the two sister chromatids separate at the centromere and segregate to opposite poles). Little is known about the causes of nondisjunction in human oocytes, but there are data suggesting that the improper alignment of chromosomes on the spindle, the failure of spindle proteins to pull homologous chromosomes to opposite poles and the premature resolution of chiasmata may be involved. a To whom correspondence should be addressed. Tel.: +33-141-14-74-17; Fax: +33-1-46-26-94-34; E-mail: mplachot@ promptomail.com
0143-4004/03/$–see front matter
FREQUENCY OF CHROMOSOME ABNORMALITIES IN HUMAN OOCYTES Two techniques are commonly used to evaluate the rate of non-disjunction in female meiosis: karyotyping by conventional cytogenetics with, in a few studies, R-banding, and FISH with various numbers of chromosome-specific probes.
Karyotype To date, almost 5000 oocytes have been analysed by conventional cytogenetics. Pooled data from previous studies shows that about 20 per cent have chromosomal abnormalities (for review, Plachot, 1997). Pellestor et al. (2002) reported the most extensive cytogenetic study to date, for which both a gradual fixation technique and an R-banding procedure were used. They karyotyped 1397 oocytes, 1088 of which (77.9 per cent) 2003 Elsevier Ltd. All rights reserved.
Plachot: Genetic Analysis of the Oocyte
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Table 1. Distribution of chromosome anomalies in 1397 oocytes Chromosome abnormalities
No. of oocytes
No. of hypohaploidies No. of hyperhaploidies No. of complex/extreme aneuploidies Total aneuploidies
75 (5.4%) 57 (4.1%) 19 (1.3%) 151 (10.8%)
No. of diploidies No. of tetraploidies No. of sets of chromatids alone No. of structural abnormalities Total abnormalities
75 (5.4%) 1 (0.07%) 53 (3.8%) 29 (2.1%) 309 (22.1%)
Adapted from Pellestor et al. (2002)
were normal (23, X). The overall frequency of chromosomal abnormalities was therefore 22.1 per cent (Table 1), similar to the mean incidence reported in published studies. Aneuploidy was observed in 10.8 per cent of oocytes: • 1.9 per cent with loss of a whole chromosome or chromosomes • 3.4 per cent with the missing chromosome or chromosomes replaced by single chromatids • 1.6 per cent with an extra whole chromosome or chromosomes • 2.5 per cent with an extra chromatid or chromatids In addition, 1.4 per cent displayed complex or extreme aneuploidy (with only 13 to 18 chromosomes). It should be noted that aneuploidy is more frequently due to single chromatids than whole chromosomes. Diploidy (5.4 per cent of cases) was observed in all oocytes displaying non-extrusion of the first polar body, and in a few cases of giant oocytes. Sets of chromatids alone (3.8 per cent of cases) were present in fertilized oocytes displaying two polar bodies and that failed to form pronuclei. In these oocytes, sperm DNA showed premature chromosome condensation. Structural abnormalities were observed in 2.1 per cent of cases and involved chromosome and chromatid breaks, deletions and acentric fragments. Given the short size and curly shape of metaphase II chromosomes, the rate of structural abnormalities is probably underestimated. Given the particular source of oocytes studied—those not fertilized by IVF—any extrapolation to the total follicular oocyte population remains hazardous. Analysis of a small number of uninseminated oocytes showed, however, that the incidence of chromosomal abnormalities was similar to that of oocytes remaining unfertilized in vitro, suggesting that gametic selection against aneuploid oocytes is probably weak or non-existent (Tarin, Gomez and Pellicer, 1991). All chromosome groups displayed aneuploidy. In comparisons of observed and estimated frequencies of aneuploidy, 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. No significant difference in the rate of chromosomal abnormalities was associated with the IVF indication in this study. Other conclusions have been drawn by several groups. De Sutter et al. (1991) observed a difference in oocyte abnormality frequency between male infertility (58 per cent normal) and non-male infertility groups (37 per cent normal), suggesting that a lack of fertilization with normal sperm may be due to oocyte factors. It is, however, still unclear to what extent genetic constitution is involved. Similar conclusions were drawn by Pellestor and Se`le (1988), who reported a higher frequency of aneuploidy in idiopathic infertility (22.5 per cent) than in male infertility (10.2 per cent), and by Selva et al. (1991), who observed more abnormalities in tubal (60 per cent) and idiopathic infertility (52 per cent) than in male infertility (34 per cent). We carried out a cytogenetic–cytological study to obtain baseline data on the status of oocytes that were not fertilized in IVF (for female infertility) or ICSI (for male infertility) procedures (Plachot, 2001). More diploid oocytes were observed in IVF (12.5 per cent) than in ICSI oocytes (1.7 per cent), indicating abnormal maturation in some oocytes from infertile women. The incidence of aneuploidy (23 per cent) and the frequency of oocytes with chromosome breaks (30 to 34 per cent) were similar in both groups. Similar results were reported by Wall et al. (1996), who observed no difference in the frequency of aneuploidy for IVF (37.3 per cent) and ICSI (31.6 per cent) groups. However, our results differ from those of Edirisinghe et al. (1997), who found that the frequency of diploidy was similar for ICSI (7.8 per cent) and IVF (9.7 per cent) groups.
FISH analysis FISH analyses of unfertilized oocytes subjected to IVF or ICSI have provided additional data about the chromosome status of human oocytes. Frequencies of aneuploidy ranging from 3 per cent (2 probes, X and 18; Dyban et al., 1996) to 38.4 per cent (4 probes, 1, 7, 15, X; Martini et al., 1997) have been reported. This wide range of chromosome abnormalities may be attributable to both the different chromosome-specific probes and the different numbers of probes used in the two studies. Comparisons with karyotyping are difficult, because with FISH only a few chromosome pairs are available for analysis, and it is possible to obtain results even from poor-quality chromosome spreads that would be discarded in karyotyping studies. Kuliev et al. (2002) reported the largest series of oocytes analysed in IVF patients in the context of preconception genetic diagnosis, to improve the success rate of IVF attempts in women of advanced age (mean 38.5 years). First (PB1) and second (PB2) polar bodies were analysed by FISH using
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probes specific for chromosomes 13, 16, 18, 21 and 22, as abnormalities are most commonly observed in these five chromosomes in spontaneous abortions and newborns. A total of 6733 oocytes were analysed, 52.1 per cent of which were aneuploid: 41.7 per cent originated from meiosis I and 35.1 per cent from meiosis II errors. In 27.5 per cent of cases, both meiotic errors occurred. Most errors in the first meiotic division (63.5 per cent) were chromatid errors, mainly missing chromatids. Non-disjunction of whole chromosomes was observed in only 6.4 per cent of cases. This excess of chromatid errors compared with chromosome errors is in accordance with the results of Pellestor et al. (2002). As many as 30.1 per cent of abnormal oocytes had complex errors involving several chromosomes, supporting the hypothesis that the frequency of meiotic spindle formation errors increases with age. Indeed, increasing the number of chromosome-specific probes used increased the proportion of abnormal oocytes with complex errors rather than the overall incidence of aneuploid oocytes. Discussing the results of preconception genetic analysis in terms of the limitations of the FISH technique or the benefits in terms of clinical pregnancy and abortion rates in women aged 35 and over is beyond the scope of this review. However, as stressed by the authors, on the one hand, the observed rates of anomalies should be overestimated as the average age of the women from whom the oocytes were obtained was 38.5 years (when compared with the mean age of IVF patients as a whole, 34.4 years in our centre). On the other hand, this may be an underestimate because only three-quarters of oocytes were tested by both PB1 and PB2 and only 3 or 5 pairs of chromosomes (out of 23) were analysed. In contrast to the well-established concept of female meiosis I origin of aneuploidy, the data from Kuliev et al. (2002) show that the observed errors originate from both meiosis I and II, and that testing of both PB1 and PB2 is required for preconception genetic analysis. The chromosomes most frequently involved in meiotic error were chromosomes 21 and 22 (20.8 per cent and 18 per cent, respectively), deriving approximately equally from meiosis I and meiosis II. Chromosome 16 errors (8.6 per cent) originated predominantly in meiosis II, in contrast to chromosome 13 (10.7 per cent) and 18 (13.3 per cent) errors, deriving more frequently from meiosis I. The limitations of oocyte genetic testing are the number of chromosome pairs tested (3 to 5), the availability of FISH results (80.3 per cent in this study) and the risk of mistakes due to both false-positive and false-negative results. Multi-locus FISH, allowing the detection of specific sequences on each chromatid of chromosomes 13 and 21, helps to differentiate between FISH failure and real aneuploidy (Eckel et al., 2002). Comparative genomic hybridization, already applied for polar body analysis, allows the copy number of every chromosome to be determined in a single experiment. It should allow any form of chromosome imbalance to be detected for either research purposes or for preconception or preimplantation genetic diagnosis (Wells et al., 2002).
Placenta (2003), Vol. 24
OOCYTE DYSMORPHY AND ANEUPLOIDY Van Blerkom and Henry (1992) demonstrated that as many as half the oocytes with dysmorphic phenotypes that arise early in meiotic maturation (organelle clustering, extensive cytoplasm vesiculization, the formation of assemblages of SER saccules, and a dark granular cytoplasm) are aneuploid, mostly hypohaploid. These oocytes fail to fertilize. In contrast, the presence of numerous endocytotic vacuoles, the exclusion or retraction of organelles from portions of the cortical cytoplasm and the appearance of small, well-delineated regions of intracellular necrosis during later stages of maturation (at or after formation of the first polar body), are associated with a low frequency of aneuploidy (<15 per cent), similar to that previously reported for morphologically normal oocytes. Giant binuclear oocytes, about 30 per cent larger than normal, account for 0.3 per cent of recovered oocytes. They are diploid and contain one or two metaphase plates (46 or 223 chromosomes) and one or two polar bodies, respectively. After fertilization, they display either two or three pronuclei and are triploid. They should therefore be excluded from uterine transfer (Balakier et al., 2002). In a small series of 65 oocytes, we found that oocyte morphology had no effect on metaphase II karyotypes (Berge`re et al., 2000).
EFFECT OF MATERNAL AGE Maternal aging is an essential factor in analysis of the frequency of aneuploidy in female gametes, although the biological mechanisms underlying this phenomenon is poorly understood. Basal FSH levels, which reflect ovarian function, increase with maternal age. Cohen et al. (1999)1 found that in women aged below 40, the frequency of chromosome abnormalities in oocytes significantly increased with basal levels of FSH (37 per cent if basal FSH <8 mIU/ml and 61 per cent if basal FSH >10 mIU/ml). In women aged over 40, there was no significant difference according to basal FSH levels. Several hypotheses have been put forward to account for the mechanism of age-related aneuploidy. It has been shown that chiasma frequency declines with increasing maternal age and that the frequency of bivalents with terminal chiasmata is much higher in older oocytes. The decrease in chiasma frequency is accompanied by the production of univalents, which are responsible for nondisjunctions. Other hypotheses connect non-disjunction with spindle disturbances. It has been suggested that spindle formation is affected by challenges to oocyte metabolism or spindle precursors during the long resting period in the dictyate stage in the ovary before the resumption of maturation. 1
Presented at the 11th World Congress on In Vitro Fertilization and Human reproductive Genetics, 9–14 May 1999, Sydney, Australia
Plachot: Genetic Analysis of the Oocyte
Aging compromises the ability of the meiotic apparatus to direct balanced chromosome segregation, and this effect has been linked indirectly to suboptimal perifollicular circulation that might compromise oocyte mitochondria. Indeed, mutations in mitochondrial DNA have been observed in the oocytes of older women. It has been suggested that the transfer of a germinal vesicle from an aged oocyte into a younger ooplasm might prevent aneuploidy (Palermo, Takeuchi and Rosenwaks, 2002). INTRAFOLLICULAR INFLUENCES Van Blerkom (1996) showed that the dissolved oxygen content of follicular fluid is follicle-specific and may differ significantly between mature, preovulatory follicles within the same ovary. For women undergoing IVF, follicular oxygen content measured at aspiration generally falls into one of two ranges, <1.5 per cent or 4–6 per cent. Cytogenetic evaluation of newly retrieved, uninseminated oocytes has shown the frequency of chromosome abnormalities to be about 45 per cent for oocytes derived from follicles with low oxygen contents, and about 7 per cent for oocytes derived from follicles with higher oxygen levels. Nearly 60 per cent of the oocytes with chromosome abnormalities retrieved from hypoxic follicles displayed some degree of chromosomal scattering at metaphase II when examined in the living state by scanning laser confocal microscopy after staining with DNA-specific fluorescent probes. Anti-tubulin immunofluorescence indicated that spindle structure was disrupted in these oocytes. This demonstrates the importance of perifollicular microvasculature expansion and follicle oxygenation during follicle growth and preovulatory maturation. There is a strong association between cigarette smoking and reduced fecundity, reduced fertility, and early mean age of menopause, suggesting that smoking may impair oocyte function and viability. Zenzes, Wang and Casper (1995) assessed the effect of smoking on the meiotic maturation of oocytes. A total of 156 women undergoing IVF, classified as non-smokers (n=102), passive smokers (n=21), light smokers (<15 cigarettes/day, n=19), and heavy smokers (>15 cigarettes/day, n=14), participated in this study. Cytogenetic data from 286 apparently unfertilized oocytes showed similar proportions of haploid (normal) and aneuploid chromosome complements in all groups. In contrast, oocytes with diploid complements were more frequent among smokers. The higher frequency of oocyte diploidy in smokers, probably resulting from the prevention of first polar body extrusion, indicates meiotic immaturity. The presence of cadmium and cotinine in follicular fluid samples provides evidence that contaminants of cigarette smoke have direct access to the follicular cells and the developing oocyte. CONCLUSION The cytogenetic investigation of human oocytes has shown that univalent and whole chromosome non-disjunction contributes
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to the high incidence of aneuploidy found in human gametes. A number of mechanisms are responsible for aneuploidy because meiosis in females is sensitive to various exogenous and endogenous factors.
REFERENCES Angell RR, Ledger W, Yong EL, Harkness L & Baird DT (1991) Cytogenetic analysis of unfertilized human oocytes. Hum Reprod, 6, 568–573. Balakier H, Bouman D, Sojecki A, Librach C & Squire J (2002) Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum Reprod, 17, 2394–2401. Berge`re M, Selva J, Plachot M, Bastit P, Guthauser B & ISFR (2000) ICSI failures: the respective influence of the oocyte morphology and of the technical parameters of ICSI. Hum Reprod, 15, Abstract Book 1, 116. De Sutter P, Dhont M, Vanluchene E & Vandekerckhove D (1991) Correlations between follicular fluid steroid analysis and maturity and cytogenetic analysis of human oocytes that remained unfertilized after in vitro fertilization. Fertil Steril, 55, 958–963. Dyban A, Freidine M, Severova E, Cieslak J, Ivakhnenko V & Verlinsky Y (1996) Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization. J Ass Reprod Genet, 13, 73–78. Edirisinghe WR, Murch A, Junk S & Yovich JL (1997) Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double blind study. Hum Reprod, 12, 2784–2791. Eckel H, Stumm M, Wieacker P & Kleinstein J (2002) Multi-locus FISH is a highly reliable method for non-disjunction studies in human oocytes. Hum Reprod, 17, Abstract book 1, 190. Kuliev A, Cieslak J, Ilkevitch Y & Verlinsky Y (2002) Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis for age-related aneuploidies. RBM Online, 6, 54–59. Martini E, Flaherty SP, Swann NJ, Payne D & Matthews CD (1997) Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes. Hum Reprod, 12, 2011–2018. Palermo GD, Takeuchi T & Rosenwaks Z (2002) Technical approaches to correction of oocyte aneuploidy. Hum Reprod, 17, 2165–2173. Pellestor F & Se`le B (1988) Assessment of aneuploidy in the human female by using cytogenetics of IVF failures. Am J Hum Genet, 42, 274–283. Pellestor F, Andre´o B, Arnal F, Humeau C & Demaille J (2002) 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. Hum Reprod, 17, 2134–2145. Plachot M (1997) The human oocyte. Genetic aspects. Ann Genet, 40, 115–120. Plachot M (2001) Chromosomal abnormalities in oocytes. Molecular and Cellular Endocrinology, 183, S59. Selva J, Martin-Pont B, Hugues JN, Rince P, Fillion C, Herve´ F, Tamboise A & Tamboise F (1991) Cytogenetic study of human oocytes uncleaved after in vitro fertilization. Hum Reprod, 6, 709–713. Tarin JJ, Gomez E & Pellicer A (1991) Chromosome anomalies in human oocytes in vitro. Fertil Steril, 55, 964–969. Van Blerkom J & Henry G (1992) Oocyte dysmorphism and aneuploidy in meiotically mature human oocytes after ovarian stimulation. Hum Reprod, 7, 379–390. Van Blerkom J (1996) The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte. J Soc Gyn Invest, 3, 1–11. Wall MB, Marks K, Smith TA, Gearon CM & Muggleton-Harris AL (1996) Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected “failedfertilized” human oocytes. Hum Reprod, 11, 2230–2238. Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JDA & Munne´ S (2002) First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril, 78, 543–549. Zenzes MT, Wang P & Casper RF (1995) Cigarette smoking may affect meiotic maturation of human oocytes. Hum Reprod, 10, 3213–3217.
Genetic Analysis of the Oocyte—A Review M. Plachot a CHI Jean Rostand, Laboratoire de Fecondation in vitro, 141 Grande Rue, 92311 Se`vres Cedex, France Paper accepted 28 May 2003
Chromosome number abnormalities are remarkably common in human reproduction. Most are caused by chromosomal non-disjunction and premature chromatid separation in oocyte meiosis I. Pooled data from previous studies showed that one in five oocytes that failed to fertilize after in vitro insemination was abnormal when analysed by conventional cytogenetics. Preconception genetic diagnosis, carried out on the first and second polar bodies by FISH, using 5 chromosome-specific probes (13, 16, 18, 21 and 22), showed that the rate of aneuploidy is higher in women aged 35 or over (52.1 per cent). Oocyte dysmorphy seems to have little effect on the rate of aneuploidy except for giant oocytes, which are usually diploid and may cause triploidy after fertilization. Intra- and extrafollicular influences (perifollicular microvasculature, oxygenation, the presence of residues from cigarette smoke) may disturb maturation, leading to immaturity and aneuploidy. Thus, oocyte meiosis is very sensitive to endogenous and exogenous factors that may cause the production of oocytes with chromosomal abnormalities and therefore, of abnormal zygotes. Placenta (2003), 24, S66–S69 2003 Elsevier Ltd. All rights reserved.
Data on the chromosomal constitution of human oocytes were reported for the first time in 1978, and have been more extensively reported during the last two decades. Indeed, the development of IVF made oocytes that failed to fertilize available for cytogenetic studies (for review, Plachot, 2001; Pellestor et al., 2002). Moreover, the development of preconceptional cytogenetic diagnosis for women of advanced age made it possible to analyse thousands of polar bodies (PBs), providing indirect information on the corresponding oocytes (Kuliev et al., 2002).
In 1991, Angell et al. (1991) observed that 13 per cent of haploid metaphases were hyperhaploid but that none contained extra whole chromosomes. The extra components were a single chromatid or two single chromatids replacing a whole chromosome. The authors suggested that the chromatids arose from premature centromere division at meiosis I, this abnormality being a major cause of trisomy. Premature chromatid division may be due to the premature degradation or lack of cohesins. These proteins exert cohesion between sister chromatids and oppose the splitting force mediated by microtubules on kinetochores (for review, Pellestor et al., 2002).
MECHANISMS OF NON-DISJUNCTION IN MEIOSIS IN HUMAN FEMALES Meiosis is the process by which a diploid cell (containing two copies of each pair of chromosomes) produces a haploid gamete containing a single copy of each chromosome. Meiotic non-disjunction is defined as the unbalanced segregation of whole chromosomes leading to hypo or hyperhaploidy. It may occur at meiosis I (when homologous chromosomes segregate to opposite poles) or at meiosis II (when the two sister chromatids separate at the centromere and segregate to opposite poles). Little is known about the causes of nondisjunction in human oocytes, but there are data suggesting that the improper alignment of chromosomes on the spindle, the failure of spindle proteins to pull homologous chromosomes to opposite poles and the premature resolution of chiasmata may be involved. a To whom correspondence should be addressed. Tel.: +33-141-14-74-17; Fax: +33-1-46-26-94-34; E-mail: mplachot@ promptomail.com
0143-4004/03/$–see front matter
FREQUENCY OF CHROMOSOME ABNORMALITIES IN HUMAN OOCYTES Two techniques are commonly used to evaluate the rate of non-disjunction in female meiosis: karyotyping by conventional cytogenetics with, in a few studies, R-banding, and FISH with various numbers of chromosome-specific probes.
Karyotype To date, almost 5000 oocytes have been analysed by conventional cytogenetics. Pooled data from previous studies shows that about 20 per cent have chromosomal abnormalities (for review, Plachot, 1997). Pellestor et al. (2002) reported the most extensive cytogenetic study to date, for which both a gradual fixation technique and an R-banding procedure were used. They karyotyped 1397 oocytes, 1088 of which (77.9 per cent) 2003 Elsevier Ltd. All rights reserved.
Plachot: Genetic Analysis of the Oocyte
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Table 1. Distribution of chromosome anomalies in 1397 oocytes Chromosome abnormalities
No. of oocytes
No. of hypohaploidies No. of hyperhaploidies No. of complex/extreme aneuploidies Total aneuploidies
75 (5.4%) 57 (4.1%) 19 (1.3%) 151 (10.8%)
No. of diploidies No. of tetraploidies No. of sets of chromatids alone No. of structural abnormalities Total abnormalities
75 (5.4%) 1 (0.07%) 53 (3.8%) 29 (2.1%) 309 (22.1%)
Adapted from Pellestor et al. (2002)
were normal (23, X). The overall frequency of chromosomal abnormalities was therefore 22.1 per cent (Table 1), similar to the mean incidence reported in published studies. Aneuploidy was observed in 10.8 per cent of oocytes: • 1.9 per cent with loss of a whole chromosome or chromosomes • 3.4 per cent with the missing chromosome or chromosomes replaced by single chromatids • 1.6 per cent with an extra whole chromosome or chromosomes • 2.5 per cent with an extra chromatid or chromatids In addition, 1.4 per cent displayed complex or extreme aneuploidy (with only 13 to 18 chromosomes). It should be noted that aneuploidy is more frequently due to single chromatids than whole chromosomes. Diploidy (5.4 per cent of cases) was observed in all oocytes displaying non-extrusion of the first polar body, and in a few cases of giant oocytes. Sets of chromatids alone (3.8 per cent of cases) were present in fertilized oocytes displaying two polar bodies and that failed to form pronuclei. In these oocytes, sperm DNA showed premature chromosome condensation. Structural abnormalities were observed in 2.1 per cent of cases and involved chromosome and chromatid breaks, deletions and acentric fragments. Given the short size and curly shape of metaphase II chromosomes, the rate of structural abnormalities is probably underestimated. Given the particular source of oocytes studied—those not fertilized by IVF—any extrapolation to the total follicular oocyte population remains hazardous. Analysis of a small number of uninseminated oocytes showed, however, that the incidence of chromosomal abnormalities was similar to that of oocytes remaining unfertilized in vitro, suggesting that gametic selection against aneuploid oocytes is probably weak or non-existent (Tarin, Gomez and Pellicer, 1991). All chromosome groups displayed aneuploidy. In comparisons of observed and estimated frequencies of aneuploidy, 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. No significant difference in the rate of chromosomal abnormalities was associated with the IVF indication in this study. Other conclusions have been drawn by several groups. De Sutter et al. (1991) observed a difference in oocyte abnormality frequency between male infertility (58 per cent normal) and non-male infertility groups (37 per cent normal), suggesting that a lack of fertilization with normal sperm may be due to oocyte factors. It is, however, still unclear to what extent genetic constitution is involved. Similar conclusions were drawn by Pellestor and Se`le (1988), who reported a higher frequency of aneuploidy in idiopathic infertility (22.5 per cent) than in male infertility (10.2 per cent), and by Selva et al. (1991), who observed more abnormalities in tubal (60 per cent) and idiopathic infertility (52 per cent) than in male infertility (34 per cent). We carried out a cytogenetic–cytological study to obtain baseline data on the status of oocytes that were not fertilized in IVF (for female infertility) or ICSI (for male infertility) procedures (Plachot, 2001). More diploid oocytes were observed in IVF (12.5 per cent) than in ICSI oocytes (1.7 per cent), indicating abnormal maturation in some oocytes from infertile women. The incidence of aneuploidy (23 per cent) and the frequency of oocytes with chromosome breaks (30 to 34 per cent) were similar in both groups. Similar results were reported by Wall et al. (1996), who observed no difference in the frequency of aneuploidy for IVF (37.3 per cent) and ICSI (31.6 per cent) groups. However, our results differ from those of Edirisinghe et al. (1997), who found that the frequency of diploidy was similar for ICSI (7.8 per cent) and IVF (9.7 per cent) groups.
FISH analysis FISH analyses of unfertilized oocytes subjected to IVF or ICSI have provided additional data about the chromosome status of human oocytes. Frequencies of aneuploidy ranging from 3 per cent (2 probes, X and 18; Dyban et al., 1996) to 38.4 per cent (4 probes, 1, 7, 15, X; Martini et al., 1997) have been reported. This wide range of chromosome abnormalities may be attributable to both the different chromosome-specific probes and the different numbers of probes used in the two studies. Comparisons with karyotyping are difficult, because with FISH only a few chromosome pairs are available for analysis, and it is possible to obtain results even from poor-quality chromosome spreads that would be discarded in karyotyping studies. Kuliev et al. (2002) reported the largest series of oocytes analysed in IVF patients in the context of preconception genetic diagnosis, to improve the success rate of IVF attempts in women of advanced age (mean 38.5 years). First (PB1) and second (PB2) polar bodies were analysed by FISH using
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probes specific for chromosomes 13, 16, 18, 21 and 22, as abnormalities are most commonly observed in these five chromosomes in spontaneous abortions and newborns. A total of 6733 oocytes were analysed, 52.1 per cent of which were aneuploid: 41.7 per cent originated from meiosis I and 35.1 per cent from meiosis II errors. In 27.5 per cent of cases, both meiotic errors occurred. Most errors in the first meiotic division (63.5 per cent) were chromatid errors, mainly missing chromatids. Non-disjunction of whole chromosomes was observed in only 6.4 per cent of cases. This excess of chromatid errors compared with chromosome errors is in accordance with the results of Pellestor et al. (2002). As many as 30.1 per cent of abnormal oocytes had complex errors involving several chromosomes, supporting the hypothesis that the frequency of meiotic spindle formation errors increases with age. Indeed, increasing the number of chromosome-specific probes used increased the proportion of abnormal oocytes with complex errors rather than the overall incidence of aneuploid oocytes. Discussing the results of preconception genetic analysis in terms of the limitations of the FISH technique or the benefits in terms of clinical pregnancy and abortion rates in women aged 35 and over is beyond the scope of this review. However, as stressed by the authors, on the one hand, the observed rates of anomalies should be overestimated as the average age of the women from whom the oocytes were obtained was 38.5 years (when compared with the mean age of IVF patients as a whole, 34.4 years in our centre). On the other hand, this may be an underestimate because only three-quarters of oocytes were tested by both PB1 and PB2 and only 3 or 5 pairs of chromosomes (out of 23) were analysed. In contrast to the well-established concept of female meiosis I origin of aneuploidy, the data from Kuliev et al. (2002) show that the observed errors originate from both meiosis I and II, and that testing of both PB1 and PB2 is required for preconception genetic analysis. The chromosomes most frequently involved in meiotic error were chromosomes 21 and 22 (20.8 per cent and 18 per cent, respectively), deriving approximately equally from meiosis I and meiosis II. Chromosome 16 errors (8.6 per cent) originated predominantly in meiosis II, in contrast to chromosome 13 (10.7 per cent) and 18 (13.3 per cent) errors, deriving more frequently from meiosis I. The limitations of oocyte genetic testing are the number of chromosome pairs tested (3 to 5), the availability of FISH results (80.3 per cent in this study) and the risk of mistakes due to both false-positive and false-negative results. Multi-locus FISH, allowing the detection of specific sequences on each chromatid of chromosomes 13 and 21, helps to differentiate between FISH failure and real aneuploidy (Eckel et al., 2002). Comparative genomic hybridization, already applied for polar body analysis, allows the copy number of every chromosome to be determined in a single experiment. It should allow any form of chromosome imbalance to be detected for either research purposes or for preconception or preimplantation genetic diagnosis (Wells et al., 2002).
Placenta (2003), Vol. 24
OOCYTE DYSMORPHY AND ANEUPLOIDY Van Blerkom and Henry (1992) demonstrated that as many as half the oocytes with dysmorphic phenotypes that arise early in meiotic maturation (organelle clustering, extensive cytoplasm vesiculization, the formation of assemblages of SER saccules, and a dark granular cytoplasm) are aneuploid, mostly hypohaploid. These oocytes fail to fertilize. In contrast, the presence of numerous endocytotic vacuoles, the exclusion or retraction of organelles from portions of the cortical cytoplasm and the appearance of small, well-delineated regions of intracellular necrosis during later stages of maturation (at or after formation of the first polar body), are associated with a low frequency of aneuploidy (<15 per cent), similar to that previously reported for morphologically normal oocytes. Giant binuclear oocytes, about 30 per cent larger than normal, account for 0.3 per cent of recovered oocytes. They are diploid and contain one or two metaphase plates (46 or 223 chromosomes) and one or two polar bodies, respectively. After fertilization, they display either two or three pronuclei and are triploid. They should therefore be excluded from uterine transfer (Balakier et al., 2002). In a small series of 65 oocytes, we found that oocyte morphology had no effect on metaphase II karyotypes (Berge`re et al., 2000).
EFFECT OF MATERNAL AGE Maternal aging is an essential factor in analysis of the frequency of aneuploidy in female gametes, although the biological mechanisms underlying this phenomenon is poorly understood. Basal FSH levels, which reflect ovarian function, increase with maternal age. Cohen et al. (1999)1 found that in women aged below 40, the frequency of chromosome abnormalities in oocytes significantly increased with basal levels of FSH (37 per cent if basal FSH <8 mIU/ml and 61 per cent if basal FSH >10 mIU/ml). In women aged over 40, there was no significant difference according to basal FSH levels. Several hypotheses have been put forward to account for the mechanism of age-related aneuploidy. It has been shown that chiasma frequency declines with increasing maternal age and that the frequency of bivalents with terminal chiasmata is much higher in older oocytes. The decrease in chiasma frequency is accompanied by the production of univalents, which are responsible for nondisjunctions. Other hypotheses connect non-disjunction with spindle disturbances. It has been suggested that spindle formation is affected by challenges to oocyte metabolism or spindle precursors during the long resting period in the dictyate stage in the ovary before the resumption of maturation. 1
Presented at the 11th World Congress on In Vitro Fertilization and Human reproductive Genetics, 9–14 May 1999, Sydney, Australia
Plachot: Genetic Analysis of the Oocyte
Aging compromises the ability of the meiotic apparatus to direct balanced chromosome segregation, and this effect has been linked indirectly to suboptimal perifollicular circulation that might compromise oocyte mitochondria. Indeed, mutations in mitochondrial DNA have been observed in the oocytes of older women. It has been suggested that the transfer of a germinal vesicle from an aged oocyte into a younger ooplasm might prevent aneuploidy (Palermo, Takeuchi and Rosenwaks, 2002). INTRAFOLLICULAR INFLUENCES Van Blerkom (1996) showed that the dissolved oxygen content of follicular fluid is follicle-specific and may differ significantly between mature, preovulatory follicles within the same ovary. For women undergoing IVF, follicular oxygen content measured at aspiration generally falls into one of two ranges, <1.5 per cent or 4–6 per cent. Cytogenetic evaluation of newly retrieved, uninseminated oocytes has shown the frequency of chromosome abnormalities to be about 45 per cent for oocytes derived from follicles with low oxygen contents, and about 7 per cent for oocytes derived from follicles with higher oxygen levels. Nearly 60 per cent of the oocytes with chromosome abnormalities retrieved from hypoxic follicles displayed some degree of chromosomal scattering at metaphase II when examined in the living state by scanning laser confocal microscopy after staining with DNA-specific fluorescent probes. Anti-tubulin immunofluorescence indicated that spindle structure was disrupted in these oocytes. This demonstrates the importance of perifollicular microvasculature expansion and follicle oxygenation during follicle growth and preovulatory maturation. There is a strong association between cigarette smoking and reduced fecundity, reduced fertility, and early mean age of menopause, suggesting that smoking may impair oocyte function and viability. Zenzes, Wang and Casper (1995) assessed the effect of smoking on the meiotic maturation of oocytes. A total of 156 women undergoing IVF, classified as non-smokers (n=102), passive smokers (n=21), light smokers (<15 cigarettes/day, n=19), and heavy smokers (>15 cigarettes/day, n=14), participated in this study. Cytogenetic data from 286 apparently unfertilized oocytes showed similar proportions of haploid (normal) and aneuploid chromosome complements in all groups. In contrast, oocytes with diploid complements were more frequent among smokers. The higher frequency of oocyte diploidy in smokers, probably resulting from the prevention of first polar body extrusion, indicates meiotic immaturity. The presence of cadmium and cotinine in follicular fluid samples provides evidence that contaminants of cigarette smoke have direct access to the follicular cells and the developing oocyte. CONCLUSION The cytogenetic investigation of human oocytes has shown that univalent and whole chromosome non-disjunction contributes
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to the high incidence of aneuploidy found in human gametes. A number of mechanisms are responsible for aneuploidy because meiosis in females is sensitive to various exogenous and endogenous factors.
REFERENCES Angell RR, Ledger W, Yong EL, Harkness L & Baird DT (1991) Cytogenetic analysis of unfertilized human oocytes. Hum Reprod, 6, 568–573. Balakier H, Bouman D, Sojecki A, Librach C & Squire J (2002) Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum Reprod, 17, 2394–2401. Berge`re M, Selva J, Plachot M, Bastit P, Guthauser B & ISFR (2000) ICSI failures: the respective influence of the oocyte morphology and of the technical parameters of ICSI. Hum Reprod, 15, Abstract Book 1, 116. De Sutter P, Dhont M, Vanluchene E & Vandekerckhove D (1991) Correlations between follicular fluid steroid analysis and maturity and cytogenetic analysis of human oocytes that remained unfertilized after in vitro fertilization. Fertil Steril, 55, 958–963. Dyban A, Freidine M, Severova E, Cieslak J, Ivakhnenko V & Verlinsky Y (1996) Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization. J Ass Reprod Genet, 13, 73–78. Edirisinghe WR, Murch A, Junk S & Yovich JL (1997) Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double blind study. Hum Reprod, 12, 2784–2791. Eckel H, Stumm M, Wieacker P & Kleinstein J (2002) Multi-locus FISH is a highly reliable method for non-disjunction studies in human oocytes. Hum Reprod, 17, Abstract book 1, 190. Kuliev A, Cieslak J, Ilkevitch Y & Verlinsky Y (2002) Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis for age-related aneuploidies. RBM Online, 6, 54–59. Martini E, Flaherty SP, Swann NJ, Payne D & Matthews CD (1997) Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes. Hum Reprod, 12, 2011–2018. Palermo GD, Takeuchi T & Rosenwaks Z (2002) Technical approaches to correction of oocyte aneuploidy. Hum Reprod, 17, 2165–2173. Pellestor F & Se`le B (1988) Assessment of aneuploidy in the human female by using cytogenetics of IVF failures. Am J Hum Genet, 42, 274–283. Pellestor F, Andre´o B, Arnal F, Humeau C & Demaille J (2002) 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. Hum Reprod, 17, 2134–2145. Plachot M (1997) The human oocyte. Genetic aspects. Ann Genet, 40, 115–120. Plachot M (2001) Chromosomal abnormalities in oocytes. Molecular and Cellular Endocrinology, 183, S59. Selva J, Martin-Pont B, Hugues JN, Rince P, Fillion C, Herve´ F, Tamboise A & Tamboise F (1991) Cytogenetic study of human oocytes uncleaved after in vitro fertilization. Hum Reprod, 6, 709–713. Tarin JJ, Gomez E & Pellicer A (1991) Chromosome anomalies in human oocytes in vitro. Fertil Steril, 55, 964–969. Van Blerkom J & Henry G (1992) Oocyte dysmorphism and aneuploidy in meiotically mature human oocytes after ovarian stimulation. Hum Reprod, 7, 379–390. Van Blerkom J (1996) The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte. J Soc Gyn Invest, 3, 1–11. Wall MB, Marks K, Smith TA, Gearon CM & Muggleton-Harris AL (1996) Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected “failedfertilized” human oocytes. Hum Reprod, 11, 2230–2238. Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JDA & Munne´ S (2002) First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril, 78, 543–549. Zenzes MT, Wang P & Casper RF (1995) Cigarette smoking may affect meiotic maturation of human oocytes. Hum Reprod, 10, 3213–3217.