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.     

Comparison of chromosomal abnormalities in hamster egg and human sperm pronuclei

One thousand human sperm and hamster egg haploid karyotypes were analyzed at the pronuclear stage after in vitro penetration. The frequency of abnormalities in human sperm was 8.5%, with 5.2% aneuploidy and 3.3% structural abnormalities. The hamster egg complements had an abnormality rate of 3.8%, with 3.3% aneuploidy and 0.5% structural abnormalities. In both human and hamster complements, chromosome abnormalities were observed in all chromosome groups, demonstrating that all chromosomes are susceptible to nondisjunction, not just acrocentric or small chromosomes. There is an intriguing difference between the frequency of hyperhaploid and hypohaploid complements in human sperm and hamster eggs. In the human complements, 2.4% were hyperhaploid and 2.7% hypohaploid. This is very close to the theoretical 1 to 1 ratio expected from nondisjunction. The hamster egg complements had more hypohaploid (2.2%) than hyperhaploid (0.9%) complements, despite identical treatment. Higher rates of hypohaploidy are generally ascribed to artificial loss of chromosomes, but may in fact reflect a predisposition of oocytes to anaphase lag during meiosis. The frequency of abnormalities (both numerical and structural) is higher in human complements than in hamster. This may reflect an innate propensity for meiotic chromosome abnormalities in humans or may result from greater exposure of humans to mutagenic agents.     

An increased frequency of human sperm chromosomal abnormalities after radiotherapy

13 cancer patients were studied before radiotherapy (RT) and at regular intervals after RT to determine the effect of RT on chromosomal abnormalities in sperm. The men were 19-47 years old and received testicular radiation doses of 0.4-5.0 Gray. Human pronuclear sperm chromosomes were analysed after penetration of zona-pellucida-free hamster eggs. Unfortunately the hamster egg penetration rates were exceedingly low, both before and after RT and this limited the number of sperm chromosome complements which could be analysed. Before RT, the frequency of abnormal sperm chromosome complements was 0% (0/9). After RT, the majority of men were azoospermic for 24 months but complements could be analysed from 4 men. In the first 12 months the frequency of abnormalities was 13% (1/8) and at 24 months it was 13% (7/55). By 36 months after RT, most men had recovered sperm production and the frequency of abnormalities in 8 men was 21% (18/86), which is significantly higher than the rate in control donors (8.5%). For individual men the range was 6-67%, and there was a significant correlation between testicular radiation dose and the frequency of sperm chromosomal abnormalities. The frequencies of both numerical and structural abnormalities were significantly increased after RT. This is the first evidence that radiation may increase the frequency of chromosomal abnormalities in human gametes.     

Similar chromosomal abnormalities in several retinoblastomas

Summary The study of banded chromosomes of nine sporadic unilateral retinoblastomas revealed near diploid karyotypes with multiple numerical and (or) structural abnormalities in all tumors. An identical marker i(6p) was noted in cells of the modal class of six retinoblastomas. Extra copies of the short arm of chromosome 6 were observed in seven tumors: +i(6p) in 6 and +6q- in one. Less regular but repeated findings were a loss of one sex chromosome, and markers 1p+ and 17q+. The structure of these markers was not identical in different tumors. Abnormalities of chromosome 13 were not observed in tumor cells, nor in blood lymphocytes stimulated by PHA.     

In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.     

Variability of human rRNA genes: inheritance and nonrandom chromosomal distribution of structural variants of nontranscribed spacer sequences

Summary Human rRNA genes contain variable regions, one of which is located in nontranscribed spacers (NTSs) closely downstream from the 3-end of the transcribed region. This polymorphism may be detected by means of blot hybridization analysis as a set of distinct restriction fragments corresponding to this part of the rRNA genes. We have analyzed DNA of 51 individuals and found eight structural NTS variants of this region; two of these were common to all individuals analyzed, and six others were found in different combinations and with different frequencies. The copy number of each variant also differed but was not less than 10–20 copies per cell. The analysis of DNA isolated from leukocytes of the members of 11 families indicated that some of the structural variants (of the NTS region) are inherited as a single Mendelian locus. We propose that rRNA genes that belong to one particular structural variant form clusters on separate chromosomes. To test this proposition, we developed a combined method, including AgNO₃-staining of chromosomes, in situ hybridization, and DNA analysis with methylation-sensitive restrictases, and used it for study of persons who had methylated rRNA genes located on AgNO₃-negative nucleolar organizers. It was found that in three of four cases methylated genes really belonged to one structural variant. This approach may be used for detailed localization of separate classes of NTS structural variants of human rRNA genes.     

Ovarian dysgenesis in individuals with chromosomal abnormalities

Summary To understand better the pathogenesis of ovarian dysgenesis in individuals with abnormalities such as 45,X Turner syndrome, trisomy 13, and trisomy 18, we have examined microscopically the ovaries of 36 infants with a number of chromosomal abnormalities confirmed by karyotype analysis. All infants with trisomy 13, trisomy 18, triploidy, and 45,X were found to have severe ovarian dysgenesis characterized by a virtual absence of primary oocytes. The ovaries of individuals with 21 trisomy and of those with partial deletion or duplication of an autosome demonstrated variable findings, which ranged from complete absence of oocytes to a mild diminution of oocyte numbers. The results of this study suggest that the attrition of germ cells in these infants is a result of faulty meiotic pairing and that ovarian dysgenesis is a more frequent finding in children with karyotypic abnormalities than has been realized previously.Presented, in part, at the 1989 David W. Smith Morphogenesis and Malformations Conference, Madrid, 1989, and at the 1990 Southern Society for Pediatric Research Meeting, New Orleans, 1990     

Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities

The most common type of karyotype abnormality detected in infertile subjects is represented by Klinefelter's syndrome, and the most frequent non-chromosomal alteration is represented by Y chromosome long arm microdeletions. Here we report our experience and a review of the literature on sperm sex chromosome aneuploidies in these two conditions. Non mosaic 47,XXY Klinefelter patients (12 subjects) show a significantly lower percentage of normal Y-bearing sperm and slightly higher percentage of normal X-bearing sperm. Consistent with the hypothesis that 47,XXY germ cells may undergo and complete meiosis, aneuploidy rate for XX- and XY-disomies is also increased with respect to controls, whereas the percentage of YY-disomies is normal. Aneuploidy rates in men with mosaic 47,XXY/46,XY (11 subjects) are lower than those observed in men with non-mosaic Klinefelter's syndrome, and only the frequency of XY-disomic sperm is significantly higher with respect to controls. Although the great majority of children born by intracytoplasmic sperm injection from Klinefelter subjects are chromosomally normal, the risk of producing offspring with chromosome aneuploidies is significant. Men with Y chromosome microdeletions (14 subjects) showed a reduction of normal Y-bearing sperm, and an increase in nullisomic and XY-disomic sperm, suggesting an instability of the deleted Y chromosome causing its loss in germ cells, and meiotic alterations leading to XY non-disjunction. Intracytoplasmic injection of sperm from Y-deleted men will therefore transmit the deletion to male children, and therefore the spermatogenic impairment, but raises also concerns of generating 45,X and 47,XXY embryos.     

Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities

Human embryonic stem cells (hESC) have great potential in regenerative medicine, provided that culture systems are established that maintain genomic integrity. Here we describe a comparison of the effects of culture in either physiologic oxygen (2%) or room oxygen (21%) on the hESC lines, H1, H9, and RH1. Physiologic oxygen enabled an average sixfold increase in clone recovery across the hESC lines tested (p < 0.001). FACS analysis showed that cells cultured in physiologic oxygen were significantly smaller and less granular. No significant changes had occurred in levels of SSEA4, SSEA1, TRA-1-60, or TRA-1-81. While karyotypic normalcy was maintained in both H1 and H9, the frequency of spontaneous chromosomal aberrations was significantly increased in room oxygen. This increase was not observed in physiologic oxygen. These results clearly demonstrate that physiologic oxygen culture conditions are indispensable for robust hES clone recovery and may enhance the isolation of novel hES lines and transgenic clones.     

Somatic chromosomal abnormalities in infertile men and women

Infertility--the inability to achieve conception or sustain a pregnancy through to live birth--is very common and affects about 15% of couples. While chromosomal or genetic abnormalities associated with azoospermia, severe oligozoospermia or primary ovarian failure were of no importance for reproduction prior to the era of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), advances in assisted reproductive techniques (ART) now enable many infertile couples to have children. These developments have raised the question of the genetic consequences of ICSI: concerns of the potential harm of the invasive procedure and concerns about the genetic risk. The infertile male and female definitely have an increased risk to carry a chromosomal abnormality. Detection of such an abnormality is of fundamental importance for the diagnosis of infertility, the following treatment, the evaluation of the risk for the future child and the appropriate management of the pregnancy to be obtained. Therefore, cytogenetic screening of both partners is mandatory prior to any type of ART. The present review is based on several surveys on male and female infertility and analyzes the types and frequencies of the different reported chromosome abnormalities according to the type of impairment of spermatogenesis and the type of treatment planned or performed. With regard to assisted reproductive techniques (especially ICSI) the main types of chromosomal abnormalities are discussed and their potential risks for ICSI. If available, reported cases of performed ICSI and its outcome are presented. The detection of an abnormal karyotype should lead to comprehensive genetic counselling, which should include all well-known information about the individual type of anomaly, its clinical relevance, its possible inheritance, the genetic risk of unbalanced offspring, and the possibilities of prenatal diagnosis. Only this proceeding allows at-risk couples to make an informed decision regarding whether or not to proceed with ART. These decisions can be made only when both partners have clearly understood the genetic risks and possible consequences when ART is used.     

Condensin dysfunction in human cells induces nonrandom chromosomal breaks in anaphase,with distinct patterns for both unique and repeated genomic regions

Condensin complexes are essential for chromosome condensation and segregation in mitosis, while condensin dysfunction, among other pathways leading to chromosomal bridging in mitosis, may play a role in tumor genomic instability, including recently discovered chromotripsis. To characterize potential double-strand breaks specifically occurring in late anaphase, human chromosomes depleted of condensin were analyzed by γ-H2AX ChIP followed by high-throughput sequencing (ChIP-seq). In condensin-depleted cells, the nonrepeated parts of the genome were shown to contain distinct γ-H2AX enrichment zones 75% of which overlapped with known hemizygous deletions in cancers. Furthermore, some tandemly repeated DNA sequences, analyzed separately from the rest of the genome, showed significant γ-H2AX enrichment in condensin-depleted anaphases. The most commonly occurring targets of such enrichment included simple repeats, centromeric satellites, and rDNA. The two latter categories indicate that acrocentric human chromosomes are especially susceptible to breaks upon condensin deficiency. The genomic regions that are specifically destabilized upon condensin dysfunction may constitute a condensin-specific chromosome destabilization pattern.     

Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.     

The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors

T cells express either of the two forms of antigen-specific receptors, the alpha/beta and gamma/delta heterodimers. Their structure closely resembles that of immunoglobulins, and the variable part of the receptor molecule is created by somatic assembly of variable, diversity, and joining regions. The genetic structure of T cell receptor (TCR) genes and their rearrangement in T cell development have been elucidated in great detail in recent years. The human genes for the gamma and beta subunits are located on the short and long arms of chromosome 7, respectively, whereas the delta- and alpha-chain genes are located in tandem on the centromeric half of the long arm of chromosome 14. Expression of either alpha/beta or gamma/delta TCR complexes on T cells in the developing thymus is likely to proceed in an ordered fashion and results in the appearance of distinct T cell subpopulations. The process of DNA rearrangements required for the generation of functional variable region genes also predisposes lymphoid cells to aberrant DNA rearrangements, which can be detected as chromosomal abnormalities such as translocations and inversions. Molecular analysis of such aberrant rearrangements has shown that rearranging loci are fused to loci unrelated to antigen receptor genes. Furthermore, the breakpoint structures represent nonproductive intermediates in the hierarchy of physiological rearrangements. Accordingly, T cell tumors arising early in T cell development often carry chromosomal abnormalities involving the delta-chain locus, whereas tumors generated later in T cell development tend to show aberrations in the alpha-chain gene. This pattern seems to reflect the stage-specific accessibility of TCR loci for rearrangement by the recombinase machinery. This enzyme is guided by specific recombination signals that can sometimes also be found at the site of breakage on the participating locus in chromosomal abnormalities. Although some features of the mechanism of aberrant rearrangements are known, their biological consequences are less well understood. However, molecular analysis of the mechanism of chromosomal aberrations in T cell tumors suggests that their biological consequences may vary. Firm evidence for the pathogenic significance is missing for most of these lesions. This provides a challenge to molecular immunology to determine how chromosomal abnormalities are involved in tumor pathogenesis.     

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.     

Mechanisms and consequences of paternally-transmitted chromosomal abnormalities

Paternally-transmitted chromosomal damage has been associated with pregnancy loss, developmental and morphological defects, infant mortality, infertility, and genetic diseases in the offspring, including cancer. There is epidemiological evidence linking paternal exposure to occupational or environmental agents with an increased risk of abnormal reproductive outcomes. There is also a large body of literature on germ cell mutagenesis in rodents showing that treatment of male germ cells with mutagens has dramatic consequences on reproduction, producing effects such as those observed in human epidemiological studies. However, we know very little about the etiology, transmission, and early embryonic consequences of paternally-derived chromosomal abnormalities. The available evidence suggests that: 1) there are distinct patterns of germ cell-stage differences in the sensitivity of induction of transmissible genetic damage, with male postmeiotic cells being the most sensitive; 2) cytogenetic abnormalities at first metaphase after fertilization are critical intermediates between paternal exposure and abnormal reproductive outcomes; and 3) there are maternal susceptibility factors that may have profound effects on the amount of sperm DNA damage that is converted into chromosomal aberrations in the zygote and that directly affect the risk for abnormal reproductive outcomes.     

Detection of chromosomal abnormalities using cordocentesis]

Four cases of cytogenetic prenatal diagnosis of fetuses with chromosomal aberrations are presented: (1) the Patau syndrome; (2) and (4) the Down syndrome; (3) the Klinefelter syndrome. Cordocentesis has been shown to be expedient for rapid and accurate determination of fetus karyotype. Indicative for cytogenetic examination were ultrasonic data, maternal age, the values of AFP, HGG and nonconjugated estreol in maternal serum. Comparison of ultrasonic examination of fetuses with the data on abortus autotopsia was undertaken. The results demonstrate importance of ultrasonic, cytogenetic, biochemical and morphological research in prenatal malformation diagnosis.