Frequency and distribution of aneuploidy in human female gametes

Summary During the past 6 years, 14 cytogenetic studies on human oocytes recovered during in vitro fertilization procedures have been published; they report contradictory results. The present survey has pooled the more than 1500 oocyte chromosome complements examined to date, in order to determine generalized trends in chromosomal abnormalities of female gametes. The overall frequency of abnormalities in mature oocytes is 24.0% with a large majority of aneuploidies (22.8%) over structural aberrations (1.2%), which could be explained by the difficulty in the detection of structural abnormalities in oocyte chromosome sets. An analysis of the distribution of non-disjunction among all chromosomes was also performed. In the A, C, D, and especially in the G groups, there is a significant difference between the observed non-disjunction and the frequencies expected from an equal partitioning of non-disjunction among all chromosomes. These data are discussed with reference to the differences obtained from cytogenetic studies on human sperm and from investigations on spontaneous abortion.     

Frequency and distribution of chromosome abnormalities in human oocytes

It was previously shown that more than half of the human oocytes obtained from IVF patients of advanced reproductive age are aneuploid, due to meiosis I and meiosis II errors. The present paper further confirms that 61.8% of the oocytes tested by fluorescent probes specific for chromosomes 13, 16, 18, 21 and 22 are abnormal, representing predominantly chromatid errors, which are the major source of aneuploidy in the resulting embryos. Almost half of the oocytes with meiosis I errors (49.3%) are prone to sequential meiosis II errors, which may lead to aneuploidy rescue in 30.8% of the cases. Half of the detected aneuploidies (49.8%) are of complex nature with involvement of two or more chromosomes, or the same chromosome in both meiotic divisions. The aneuploidy rates for individual chromosomes are different, with a higher prevalence of chromosome 21 and 22 errors. The origin of aneuploidy for the individual chromosomes is also not random, with chromosome 16 and 22 errors originating more frequently in meiosis II, and chromosome 18, 13 and 21 errors in meiosis I. There is an age dependence not only for the overall frequency of aneuploidies, but also for each chromosome error, aneuploidies originating from meiosis I, meiosis II, and both meiosis I and meiosis II errors, as well as for different types of aneuploidies. The data further suggest the practical relevance of oocyte aneuploidy testing for detection and avoidance from transfer of the embryos deriving from aneuploid oocytes, which should contribute significantly to the pregnancy outcomes of IVF patients of advanced reproduction age.     

The incidence of aneuploidy in human oocytes assessed by conventional cytogenetic analysis

Human oocytes failing to fertilize during assisted reproduction are an important source of information for assessing incidence and causal mechanisms of maternal aneuploidy. This review describes the techniques of conventional oocyte chromosome analysis and evaluates the results of 59 studies comprising a total of>10,000 female gametes. The mean rate of aneuploidy (hypohaploidy + hyperhaploidy) amounts to approximately 20%, but this incidence is reduced as soon as possible artifacts introduced by the fixation technique are taken into consideration. It is therefore concluded that a realistic value for numerical abnormalities arising during first meiotic division lies between 12 and 15%. All chromosome groups are affected by aneuploidy but the actually observed frequencies exceed the expected frequencies in groups D, E, and G. Two aneuploidy-causing mechanisms have been identified in human oocytes: nondisjunction, resulting in the loss or gain of whole chromosomes, and predivision, resulting in the loss or gain of single chromatids. A brief analysis including only aneuploid complements with one extra or missing chromosome/chromatid shows a slight increase in predivision (52.9%) compared with nondisjunction (47.1%). Finally, suggestions for future studies are given since, for instance, the presentation of results and the use of cytogenetic nomenclature have not been uniform.     

The origin and impact of embryonic aneuploidy

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.     

Chromosome abnormalities in the human oocyte

Aneuploidy is the most commonly occurring type of chromosome abnormality and the most significant clinically. It arises mostly due to segregation errors taking place during female meiosis and is also closely associated with advancing maternal age. Two main aneuploidy-causing mechanisms have been described: the first involves the non-disjunction of entire chromosomes and can take place during both meiotic divisions, whereas the second involves the premature division of a chromosome into its 2 sister chromatids, followed by their random segregation, upon completion of meiosis I. To elucidate the causal mechanisms of maternally derived aneuploidy and the manner with which they affect the 2 meiotic divisions, a large number of oocytes and their corresponding polar bodies have been examined. Various classical and molecular cytogenetic methods have been employed for this purpose, and valuable data have been obtained. Moreover, research into the gene expression patterns of oocytes according to maturity, maternal age, and chromosome status has provided a unique insight into the complex nature of the biological processes and genetic pathways regulating female meiosis. Findings obtained from the cytogenetic and molecular analysis of oocytes will be reviewed in this article.     

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

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

Frequency of aneuploidy related to age in porcine oocytes

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.     

Features of chromosome segregation in unfertilized human oocytes

Mechanisms of abnormal chromosomal segregation in female meiosis and factors exerting influence on unfertilized human oocytes have been analysed in vitro. The differences in results of cytological studies on the rate of aneuploidy have been reviewed. The advanced maternal age is primarily associated with disturbances in oocyte meiosis.     

Meiosis in man

The study of human meiosis impinges on clinically important areas such as infertility and aneuploidy, and an understanding of the behaviour of chromosomes in the germ line is essential for those engaged in genetic counselling. Recent advances in cytological techniques are allowing detailed observation of chromosomes in the prophase stage. Important problems such as the effects of structural and numerical chromosome abnormalities, and the relationship between abnormal meiotic chromosome behaviour and germ-cell death are being explored, while studies on mouse oocytes are beginning to shed light on the basis of the maternal age effect for aneuploidy such as in Down syndrome in man. Work at the molecular level promises to yield further advances.     

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: oligozoospermia

Recently, intracytoplasmic sperm injection (ICSI) has been extremely successful for the treatment of male infertility. However, transmission of cytogenetic defects to offspring is a great concern. There are two types of cytogenetic problems in patients seeking ICSI; one is the transmission of genetic defects from patients with constitutional chromosomal abnormalities and the second is the generation of de novo defects in infertile men. Generally about 5.1% of infertile men have chromosomal abnormalities. Among such infertile men, men with severe spermatogenesis defects, including oligozoospermia and azoospermia, are subjects for ICSI. Therefore it is very important to obtain cytogenetic information in these infertile patients. Furthermore, oligozoospermic men with a normal somatic karyotype also have increased frequencies of sperm chromosome abnormalities. Oligozoospermia is usually associated with other sperm alterations, for example oligoasthenozoospermia, oligoteratozoospemia and oligoasthenoteratozoospermia. In this review, the relationship between sperm concentration and sperm aneuploidy frequencies has been analyzed. The inverse correlation between the frequency of sperm aneuploidy and concentration has been reported in extensive studies. Especially in severe oligozoospermia, a significantly higher frequency of sex chromosome aneuploidy has been observed and this has been corroborated in recent clinical outcome data of ICSI.     

Maternal age and chromosomal abnormalities in human oocytes

Maternal ageing is the only etiological factor unequivocally associated with the occurrence of aneuploid conceptuses. Molecular studies of trisomies have demonstrated that the pattern of recombinaison was an important predisposing factor to meiotic nondisjunction. To complete this data, a large chromosomal study has been undertaken on 1,397 unfertilised human oocytes recovered from women participating in in vitro fertilization programmes. Conventional whole chromosome nondisjunction and premature chromatid separation were the major types of numerical abnormalities observed. A positive relationship was found between maternal age and these two types of nondisjunction, but the most significant correlation was observed with chromatid separation resulting in the presence of free chromatid in metaphase II oocyte. These data revealed that chromatid separation was an essential factor in the age-dependent occurrence of aneuploidy. This finding provided new insights into the mechanism of nondisjunction in female meiosis since disturbance in molecular chromatid cohesion by cohesins might be a causal mechanism predisposing to nondisjunction and involved in the maternal age effect.     

The effect of age on the frequency of sperm chromosomal abnormalities in normal men.

It has been suggested that advanced paternal age (independent of maternal age) is associated with an increased incidence of trisomy. However, studies of human liveborn offspring and of data from prenatal diagnosis have yielded conflicting results. To investigate this possible paternal age effect, we have studied sperm chromosome complements from 30 normal men of proven fertility stratified by age, with five males in each of six age categories (20-24, 25-29, 30-34, 35-39, 40-44, and 45+ years). Sperm chromosome complements were visualized after penetration of golden-hamster oocytes. A minimum of 30 complements were analyzed for each male. The analysis was performed blindly, without knowledge of the donor's age. The mean frequency of sperm chromosomal abnormalities in the individual men was 10.4% with means of 4.7% for numerical abnormalities and 6.2% for structural abnormalities. There was no relationship between age and the frequency of numerical abnormalities in sperm. Since there was a significant difference between the frequency of hyperhaploid and hypohaploid complements, these two types of numerical abnormalities were analyzed separately. There was no correlation between the frequency of hypohaploid complements and age. There was a significant negative correlation between age and the frequency of hyperhaploid complements. For structural abnormalities, there was a highly significant positive correlation with age. Thus, our results do not support the hypothesis of an increased risk of trisomy with paternal age.     

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation

Frozen-thawed mouse oocytes were artificially activated with Sr²⁺ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (>90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozenthawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0°C or 37°C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0°C without DMSO or held at 37°C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos. © 1995 wiley-Liss, Inc.     

Frequency and distribution of aneuploidy in human gametes: differences as a function of sex]

The frequency and the distribution of aneuploidies were analysed in both spermatozoa and mature oocyte. The present study has pooled 13,975 human sperm chromosome complements and 1,897 oocyte chromosome complements examined to date. The overall frequency of aneuploidy is 10% in spermatozoa and 22.4% in oocytes. Human sperm is characterized by a significant excess of hypo-haploidies and an equitable distribution of aneuploidies among all chromosome groups, whereas mature oocytes display an equal ratio of hypo-haploidies: hyper-haploidies and a high variability in the distribution of non-disjunctions; in the A, B, C and especially in D and G groups, there is a significant difference between the observed and estimated rates of non-disjunction and the frequencies expected from an equal partitioning of non-disjunctions among all chromosomes. This indicates that non-disjunction is not a random event in female meiosis, and consequently that there are differences in the meiotic process between the sexes.     

Evaluation of aneuploidy frequency for chromosomes 6 and 17 in eyelid tumours using the FISH technique

Although basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are very common skin tumours, the incidence of chromosome aneuploidy with regard to the eyelid has not been investigated. We aimed to find the frequency of chromosome 6 and 17 aneuploidies in eyelid tumours' interphase nuclei with fluorescence in situ hybridization (I-FISH) with chromosome specific DNA probes. I-FISH with chromosome 6 and 17 centromere specific DNA probes was used in the eyelids of 10 patients with BCC or SCC and the peripheral blood cells of 10 healthy donors as controls. The frequency of chromosome 6 and 17 aneuploidies was significantly higher in 7 out of 10 patients and 5 out of 10 patients, respectively, than in controls, indicating a higher frequency of aneuploidy in BCC than in SCC of the eyelid. Distribution of hybridization signals for chromosome 6 and 17 was wide ranging, indicating heterogeneity of cell populations with aneuploidy between patients. These findings indicate that acquisition of chromosome aneuploidies in eyelid tumours may have an important pathogenic role in both BCC and SCC of the eyelid area.     

Sexual immaturity and maternal age: incidence of aneuploidy and polyploidy in first-cleavage mouse embryos

The influence of maternal age on the incidence of aneuploidy and polyploidy was studied, using C57Bl/6J X CBA/Ca hybrid mice, including immature females, as gamete donors. The age of the females ranged from 3.5-4 wk (immature or prepubertal), to 10-12 wk (young adults), to 24-28 wk (aged females). Ovulation was induced with gonadotrophins, and the differential condensation of paternal and maternal chromosomes was used to elucidate the origin of chromosome abnormalities in first-division metaphase plates. The results indicated a high incidence of aneuploid oocytes in immature and older female mice, as compared to young adult females. Eggs of immature female mice underwent polyspermic fertilization more often than those of young adults and older females, and the production of diploid oocytes was more frequent in immature females than in the other age groups.     

Cytogenetic results of amniocentesis materials: incidence of abnormal karyotypes in the Turkish collaborative study

The experience on prenatal chromosome diagnosis of four Turkish centers participating in a collaborative study on 6041 genetic amniocentesis performed during a 4-8 years period were reviewed. 5887 (97.5%) patients had strong clinical indications for prenatal chromosome studies and 154 (2.5%) were referred because of maternal anxiety and a bad history of previous gestations. The main indication groups were: advanced maternal age (3197 cases), positive serum screening (2011 cases), ultrasound-identified anomaly (492 cases), previous fetus/child with chromosomal aberrations (103 cases), a history of a previous abnormal and/or mentally handicapped child (70 cases) and a parental chromosome rearrangement (14 cases). The average maternal age was 33.9 years and average gestational age was 18 weeks. A total of 179 affected fetuses were detected in this collaborative study (3%) of which 133 were unbalanced (74.3%). Among the 124 (69%) numerical aberrations, 102 (82.3%) were autosomal aneuploidies, 20 (16.1%) were gonosomal aneuploidies and 2 (1.6%) were poliploidies. Among the 55 (31%) structural aberrations, balanced translocation was the most common (63.6%) and 11 cases of inversion, four cases of unbalanced translocation, two cases of marker chromosome and three cases of other abnormalities were found. The overall culture success rate was 99.7%. Pregnancy termination that is permitted by legal authorities was accepted by 94.7% (126/133) with parents at unbalanced cytogenetic result announcement.     

Chromosomal analysis of unfertilized human oocytes

Unfertilized human oocytes were obtained from women in an in-vitro fertilization programme. The women had a mean age of 29.4 years (range 24-35 years). Chromosomal complements could be analysed in 50 oocytes. Q-banding of the chromosomes facilitated identification of individual chromosomes: 34 oocytes (68%) had the normal haploid chromosomal complement, 14 complements were hypohaploid (28%), 1 complement was hyperhaploid (2%) and 2 had structural abnormalities (4%). (One oocyte had numerical and structural abnormalities). The 16 abnormal oocytes were obtained from 15 different women. A conservative estimate of aneuploidy in this sample is 4%; however, the frequency of aneuploidy may be higher if there is a predisposition to chromosome loss during oogenesis. This study provides information on the largest series of karyotyped unfertilized human oocytes published to date.     

用FISH技术研究人类体外未受精卵的21号染色体非整倍体

采用荧光原位杂交技术,选用人类21号染色体端粒探针（21qter）,检测人类体外未受精卵的21号染色体非整倍体发生率,并比较非整倍体率与25－30岁和31－35岁这两个女性年龄组、IVF指征、超排方案之间的关系,在54个未受精卵中,正常21号单体30枚,二体16枚,三体4枚,缺体4枚,非整倍体率为44.4％（24／54）;25－30岁和31－35岁这两个年龄组、IVF指征、超排方案的患者的21号染色体非整倍体率之间的差异无显著性,卵母细胞21号染色体的非整倍性是造成体外受精失败的重要原因之一。