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.     

Chromosomal anomalies of human gametes and intrauterine selection. Research on female gametes]

The results of cytogenetical analysis of human oocytes are summarized. The chromosomal disorders in human gametes and in spontaneous abortions are comparatively analyzed.     

Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the in vivo/in vitro technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.     

Induced fusion of female gametes and embryonic cells

Investigations in which the method of induced cell fusion was used in studying the oocyte maturation, fertilization and early embryogenesis of animals have been reviewed on the basis of published data and the authors' experience. The problems of developing the method of induced cell fusion, its combination with other methods (microsurgery, cell fragmentation, etc.) and the perspectives of its use in embryology are discussed.     

Assessment of aneuploidy in the human female by using cytogenetics of IVF failures

The karyotype was determined for 201 unfertilized human oocytes recovered from 87 women participating in an in vitro fertilization program. Their mean age was 30.6 years (range 22-40 years). Thirteen oocytes did not exhibit the first polar body and were found to be in the first-metaphase stage. The remaining 188 eggs were assessed as mature and classified into two groups according to the supposed origin of fertilization failure. The overall incidence of aneuploidy was 18.6%. This rate was higher in the group of unexplained fertilization failure (22.5%) than in the group of fertilization failure due to sperm deficiency (10.2%). This may reflect a relationship between the genomic constitution of the oocyte and its fertilizability. On the other hand, no increase of aneuploidy was observed with maternal aging.     

Non-cell-autonomous small RNA silencing in Arabidopsis female gametes

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Frequency and distribution of chromosome abnormalities in human spermatozoa

This study reviews the frequency and distribution of numerical and structural chromosomal abnormalities in spermatozoa from normal men obtained by the human-hamster system and by multicolor-FISH analysis on decondensed sperm nuclei. Results from large sperm karyotyping series analyzed by chromosome banding techniques and results from multicolor FISH in sperm nuclei (of at least 10(4) spermatozoa per donor and per probe) were reviewed in order to establish baseline values of the sperm chromosome abnormalities in normal men. In karyotyping studies, the mean disomy frequency in human sperm is 0.03% for each of the autosomes, and 0.11% for the sex chromosomes, lower than those reported in sperm nuclei by FISH studies using a similar methodology (0.09% and 0.26%, respectively). Both types of studies coincide in that chromosome 21 and sex chromosomes have a greater tendency to suffer segregation errors than the rest of the autosomes. The mean incidence of diploidy, only available from multicolor FISH in sperm nuclei, is 0.19%. Inter-donor differences observed for disomy and diploidy frequencies among FISH studies of decondensed sperm nuclei using a similar methodology could reflect real differences among normal men, but they could also reflect the subjective application of the scoring criteria among laboratories. The mean frequency of structural aberrations in sperm karyotypes is 6.6%, including all chromosome types of abnormalities. Chromosome 9 shows a high susceptibility to be broken and 50% of the breakpoints are located in 9q, between the centromere and the 9qh+ region. Structural chromosome aberrations for chromosomes 1 and 9 have also been analyzed in human sperm nuclei by multicolor FISH. Unfortunately, this assay does not allow to determine the specific type of structural aberrations observed in sperm nuclei. An association between advancing donor age and increased frequency of numerical and structural chromosome abnormalities has been reported in spermatozoa of normal men.     

Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.     

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.     

Chromosome abnormalities of human gametes and intrauterine selection. Male gametes]

Man appears to be unique among mammals with respect to very high level of reproductive wastage. It seems reasonable to suppose that many early losses attribute to cytogenetical abnormalities. However, this stage of pregnancy is difficult to be studied. The investigation of gametes is one of the most reliable ways to estimate the cytogenetical disorders in early life. The results from cytogenetical analysis of human male gametes are summarized.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

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.