用荧光原位杂交技术研究人类未受精卵细胞的染色体非整倍体

目的　探讨人类未受精卵细胞非整倍体的产生机制。方法　取未受精卵细胞固定后行多色荧光原位杂交 ,分析卵细胞中 13、16、18、2 1和 2 2号染色体的核型情况。结果　 4 7%的卵细胞核型正常 ,5 3%的卵细胞为异常核型 ,其中 18%为同源染色体不分离 ,12 %为姐妹染色单体非平衡性过早分离 ,36 %为姐妹染色单体平衡性过早分离 ;在体外培养 >2 4小时的卵细胞中 ,姐妹染色单体平衡性过早分离的发生率明显高于体外培养≤ 2 4小时的卵细胞 ( P<0 .0 1)。结论　同源染色体不分离和姐妹染色单体平衡性及非平衡性过早分离这三种机制均参与了卵细胞非整倍体的产生。姐妹染色单体平衡性过早分离与体外培养时间具有相关性     

Spectral karyotyping of fresh,non-inseminated oocytes

The object of this study was to determine the mechanisms producing aneuploidy in female meiosis I by analysing the whole chromosome complement of human non-inseminated and unfertilized fresh oocytes. For this purpose, 131 fresh oocytes were obtained from 16 oocyte donors (24-48 years old). These oocytes were fixed immediately after retrieval and 47 good quality metaphases from 13 donors were analysed by spectral karyotyping to identify all 23 chromosome types. The data was divided into two maternal age groups, 24-34 (n = 31) and > or = 35 years (n = 16). More non-disjunction (13 and 25%), unbalanced predivision (10 and 44%, P < 0.01) and balanced predivision (6 and 62%, P < 0.001) events were found in the older group of oocytes. There was an increase in balanced predivision with decreasing chromosome size (P < 0.001). The present results are the first obtained in fresh oocytes where all chromosomes were specifically identified, and support previous theories that predivision of chromatids is a major factor causing aneuploidy. Previous reports with inseminated, non-fertilized oocytes fixed > or = 24 h after retrieval suffered from artefactual predivision of chromatids triggered by in-vitro ageing of oocytes.     

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.     

多色荧光原位杂交检测人卵细胞染色体非整倍体方法的建立

目的 建立多色荧光原位杂交技术检测人卵细胞染色体非整倍体的方法。方法 取试管婴儿助孕技术后未能受精成功的卵细胞，于取卵后13d固定，采用多色荧光原位杂交方法检测卵细胞13，16，18。21和22号染色体的情况。结果 正常未受精卵细胞中期染色体显示一个成对的杂交信号，每条染色单体显示一个单个信号；分裂相中多出或缺少一个成对杂交信号表明多余或缺少一条染色体；分裂相中多出或缺少一个单个信号表明多余或缺少一条染色单体；两个单个信号分离表明两条姐妹染色单体分离。结论 采用多色荧光原位杂交方法可以有效检测人卵细胞染色体非整倍体异常。     

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.     

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.     

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.     

排卵后老化对卵母细胞姐妹染色单体平衡性早分离的影响

目的探讨排卵后老化对卵母细胞姐妹染色单体平衡性早分离（balanced predivision，BP）的影响。方法分别采用荧光原位杂交和免疫细胞化学方法检测排卵后不同培养时间下小鼠成熟卵母细胞16号染色体的核型及纺锤体和染色体构象的变化。结果新鲜小鼠卵母细胞中BP的发生率为7％，培养24h、48h和72h后，卵母细胞BP发生率分别为32％、53％和6l％，明显高于新鲜卵母细胞（P〈0．01）；新鲜小鼠卵母细胞的纺锤体和染色体异常比率为9％，明显低于培养24h、48h和72h后卵母细胞的纺锤体和染色体异常率（P〈0．01）。结论BP可发生于卵母细胞排卵后老化过程中，其发生可能与纺锤体和染色体构象改变有关。     

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.     

光谱核型分析检测人未受精卵细胞非整倍体

目的 建立光谱核型分析检测人未受精卵细胞非整倍体的方法。方法 取试管婴儿术后成熟未受精卵细胞，固定后行光谱核型分析，检测卵细胞各染色体的核型情况。结果 64％的卵细胞核型正常，36％的卵细胞为非整倍体，其中22％为同源染色体不分离，14％为姐妹染色单体非平衡性过早分离。结论 光谱核型分析可用于检测卵细胞各条染色体的异常。同源染色体不分离和姐妹染色单体非平衡性过早分离均参与了卵细胞非整倍体的产生。     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

Cytogenetic analysis of human zygotes displaying three pronuclei and one polar body after intracytoplasmic sperm injection.

BACKGROUND: Digynic zygotes with three pronuclei and one polar body obtained after intracytoplasmic sperm injection (ICSI) were studied cytogenetically to elucidate the frequency and origin of chromosomal abnormalities at the earliest stage of conception. METHODS: Uncleaved, single-cell zygotes were incubated with podophyllotoxin and vinblastine and fixed by a gradual fixation air drying method. The chromosomes were stained with Giemsa. RESULTS: Twenty-two (50%) out of 44 informative zygotes revealed cytogenetic alterations, including aneuploidy (six cells, 13.6%), structural aberrations (10 cells, 22.7%) and combinations of numerical and structural abnormalities (two cells, 4.5%). In one case (2.3%), double aneuploidy or an effect of chromosomal translocation could not be distinguished and one zygote (2.3%) turned out tetraploid due to injection of a diploid spermatozoon. Two zygotes (4.5%) showed an irregular chromatid segregation between the two maternal complements. In completely analysable cells, the sex chromosome ratio XXX:XXY was 17:15. CONCLUSIONS: Digynic ICSI zygotes carry a high rate of cytogenetic abnormalities that obviously have been transmitted by the participating oocytes and spermatozoa. We also confirmed the previously reported, possibly ICSI-induced irregular oocyte chromatid segregation. The results suggest that aneuploidy in the oocytes must have been caused by predivision instead of non-disjunction.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.     

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.     

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.     

Chromosomal FISH analysis of unfertilized human oocytes and polar bodies

 The incidence of chromosomal aneuploidy was studied in 208 unfertilized metaphase II human oocytes from an in vitro fertilization program by fluorescence in situ hybridization using probes for chromosomes 18, 21, and X. Chromosome spreads were prepared by a gradual fixation–air-drying method. We obtained analyzable results from 183 oocytes and 93 polar bodies; 167 oocytes (91%) were normal, 11 (6%) were diploid, and 5 (3%) were aneuploid. Of the five aneuploid oocytes, four involved chromosome 21, and one involved the X chromosome. In this study, oocyte aneuploidy caused by both nondisjunction of bivalent chromosomes and predivision of univalent chromosomes was observed. The aneuploidy rate (9.8%) in the oocytes from women aged ≧35 years was significantly higher than that (0.7%) in those aged 23 to 34 years (P= 0.0017). Received: February 14, 2002 / Accepted: June 10, 2002     

Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induces premature separation of sister chromatids during meiosis I and aneuploidy in mouse oocytes in vitro

Recent advances in understanding some of the molecular aspects of chromosome segregation during mitosis and meiosis provide a background for investigating potential mechanisms of aneuploidy in mammalian germ cells. Numerous protein kinases and phosphatases have important functions during mitosis and meiosis. Alterations in these enzyme activities may upset the normal temporal sequence of biochemical reactions and cellular organelle modifications required for orderly chromosome segregation. Protein phosphatases 1 (PP1) and 2A (PP2A) play integral roles in regulating oocyte maturation (OM) and the metaphase–anaphase transitions. Mouse oocytes were transiently exposed invitro to different dosages (0, 0.01, 0.1, or 1.0 μg/ml) of the PP1 and PP2A phosphatase inhibitor okadaic acid (OA) during meiosis I and oocytes were cytogenetically analyzed. Significant (p < 0.05) OA dose-response increases in the frequencies of metaphase I (MI) arrested oocytes, MI oocytes with 80 chromatids instead of the normal 20 tetrads, and anaphase I–telophase I (AI–TI) oocytes with two groups of an unequal number of chromatids were found. Analysis of MII oocytes revealed significant (p < 0.05) increases in the frequencies of premature sister chromatid separation, single-unpaired chromatids, and hyperploidy. Besides showing that OA is aneugenic, these data suggest that OA-induced protein phosphatase inhibition upsets the normal kinase–phosphatase equilibrium during mouse OM, resulting in precocious removal of cohesion proteins from chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Meiosis-activating sterol protects oocytes from precocious chromosome segregation

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. RESULTS: FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. CONCLUSIONS: FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation.     

Chromosomes of oocytes failing in-vitro fertilization

Of 263 oocytes that failed to fertilize after in-vitro fertilizationand were sent for cytogenetic investigation, 179 (68.1 %) wereanalysable. More than 72.0% were normal metaphase II haploids(23,X). Hyperhaploidy, hypohaploidy and complex cases made upa total aneuploidy rate of 12.3%, while 10.1% were diploid.In addition, there were five oocytes with structural aberrationsand eight yielded chromatids only. The total chromosome aberrationrate was 29.1%. No significant difference was found betweenthe aneuploidy rate and maternal age. Here we present photographicvidence of oocytes with extra whole chromosomes and extra singlechromatids. We suggest that both predivision and nondisjunctioncontribute to the formation of trisomy in man.     

The use of first polar bodies for preimplantation diagnosis of aneuploidy

A large proportion of patients undergoing in-vitro fertilization(IVF) are aged 35 years. It has been estimated that in thisage group, 50% of embryos are chromosomally abnormal, with aneuploidybeing the major contributing factor. Since the origin of mostaneuploidies is maternal meiosis I non-disjunction, unfertilizedoocytes could be safely screened for aneuploidy by analysingtheir first polar bodies. To determine the feasibility of firstpolar body aneuploidy analysis, polar bodies were analysed byfluorescence in-situ hybridization (FISH) using probes simultaneouslyfor chromosomes X, Y, 18, 13/21 or X, Y, 18 and 16. Within 6h of retrieval, 88% showed a normal segregation involving asingle chromosome of each kind, with double-dotted hybridizationsignals, corresponding to dyads (chromosomes in metaphase Icomposed of two chromatids). The rest showed non-disjunctionof full dyads (6%), or an unbalanced pre-division of dyads (6%),which gives a segregation of one chromatid or one dyad and achromatid with the first polar body. But only 34% of polar bodiesanalysed 24 h after retrieval or later showed a normal segregation,with most of the other polar bodies showing balanced pre-division,with two separated hybridization signals for all the chromosomesanalysed. The rates of non-disjunction and unbalanced pre-divisionafter24 h in culture were similar to the rates in fresh oocytes.When both types of aneuploidy were considered together, an increaseof aneuploidy with maternal age was detected, which althoughslight, was significant (P = 0.025). Because dyads seem to undergorapid pre-division shortly after polar body retrieval, performanceof FISH aneuploidy analysis of polar bodies is therefore onlyrecommended when conducted within 6 h of their retrieval. ICSI/oocyte/pre-division/preimplantation genetic diagnosis/trisomy 16