Chromoanagenesis, the mechanisms of a genomic chaos.

Designated under the name of chromoanagenesis, the phenomena of chromothripsis, chromanasynthesis and chromoplexy constitute new types of complex rearrangements, including many genomic alterations localized on a few chromosomal regions, and whose discovery over the last decade has changed our perception about the formation of chromosomal abnormalities and their etiology. Although exhibiting specific features, these new catastrophic mechanisms generally occur within a single cell cycle and their emergence is closely linked to genomic instability. Various non-exclusive exogenous or cellular mechanisms capable of generating chromoanagenesis have been evoked. However, recent experimental data shed light on 2 major processes, which following a defect in the mitotic segregation of chromosomes, can generate a cascade of cellular events leading to chromoanagenesis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material, and the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions. In both cases, the cellular and molecular mechanisms of fragmentation, repair and transmission of damaged chromosomal material are consistent with the features of chromoanagenesis-related complex chromosomal rearrangements. In this review, we introduce each type of chromoanagenesis, and describe the experimental models that have allowed to validate the existence of chromoanagenesis events and to better understand their cellular mechanisms of formation and transmission, as well as their impact on the stability and the plasticity of the genome.

Discordant sex in monozygotic XXY/XX twins: a case report.

We report a case of discordant phenotypic sex in monozygotic twins mosaic 47,XXY/46,XX: monozygotic heterokaryotypic twins. The twins presented with cognitive and comprehension delay, behavioural and language disorders, all symptoms frequently reported in Klinefelter syndrome. Molecular zygosity analysis with several markers confirmed that the twins are in effect monozygotic (MZ). Array comparative genomic hybridization found no evidence for the implication of copy number variation in the phenotypes. Ultrasound scans of the reproductive organs revealed no abnormalities. Endocrine tests showed a low testosterone level in Twin 1 (male phenotype) and a low gonadotrophin level in Twin 2 (female phenotype) which, combined with the results from ultrasound examination, provided useful information for potentially predicting the future fertility potential of the twins. Blood karyotypes revealed the presence of a normal 46,XX cell line and an aneuploïd 47,XXY cell line in both patients. Examination of the chromosome constitutions of various tissues such as blood, buccal smear and urinary sediment not surprisingly showed different proportions for the 46,XX and 47,XXY cell lines, which most likely explains the discordant phenotypic sex and mild Klinefelter features. The most plausible underlying biological mechanism is a post-zygotic loss of the Y chromosome in an initially 47,XXY zygote. This would result in an embryo with both 46,XX and 47,XXY cells lines which could subsequently divide into two monozygotic embryos through a twinning process. The two cell lines would then be distributed differently between tissues which could result in phenotypic discordances in the twins. These observations emphasize the importance of regular paediatric evaluations to determine the optimal timing for fertility preservation measures and to detect new Klinefelter features which could appear throughout childhood in the two subjects.

Complex chromosomal rearrangements: origin and meiotic behavior.

BACKGROUND: Complex chromosomal rearrangements (CCRs) describe structural rearrangements, essentially translocations, involving at least three breakpoints on two or more chromosomes. Although they are rare in humans, their clinical identification is important since CCR carriers can display various phenotypes which include phenotypically normal subjects, infertile males and patients with mental retardation and/or congenital abnormalities. The rearrangement can be de novo or familial. The use of fluorescent in situ hybridization assays and molecular techniques for the characterization of CCRs have indicated that the rearrangements could be more complex than initially assumed. Accumulating data have revealed that the mechanisms underlying the genesis of CCRs remain elusive. METHODS: We performed a large PubMed search in order to summarize the current knowledge in this field and address important aspects of CCR formation and meiotic behavior, highlighting the complexity of these rearrangements at the chromosomal and genomic level. RESULTS: The review of published data indicates that the complexity of CCRs is becoming increasingly known, thanks to the application of more and more efficient molecular techniques. These approaches have allowed the precise sequence analysis of breakpoints and the identification of insertions, deletions, inversions and recombination events. New models have been proposed for the formation of CCRs, based on replication-based mechanisms and specific sequence elements. Their meiotic behavior has been discussed in the light of these new molecular data. CONCLUSIONS: Despite the increasing understanding of the mechanisms involved in their genesis, CCRs arise as unique, complex events for which the genetic and reproductive counseling of carriers remains a challenge.

Apoptosis and meiotic segregation in ejaculated sperm from Robertsonian translocation carrier patients.

BACKGROUND: To better understand the infertility of patients with Robertsonian translocation, the biochemical and ultrastructural apoptotic characteristics of apoptosis in the sperm of patients and fertile donors were studied. METHODS: Ejaculated sperm samples of seven Robertsonian translocation carriers and seven fertile donors were analyzed after cryopreservation. The proportion of both viable and dead spermatozoa expressing activated caspases was detected by flow cytometry through the use of different specific carboxyfluorescein-labeled caspase inhibitors. Sperm DNA fragmentation was evaluated by the TUNEL method. The percentages of intact spermatozoa or spermatozoa with ultrastructural features of apoptosis, immaturity or necrosis were estimated by electron microscopy. Meiotic segregation analysis was performed by FISH. RESULTS: Significantly lower concentration, forward motility and normal morphology of spermatozoa were found in ejaculated samples of the Robertsonian patients than fertile donors. Compared with the control group, in Robertsonian translocation carriers: (i) the caspase assays showed a significantly increased (P < 0.05) proportion of viable spermatozoa with activated poly-caspases (57.4 versus 25.8%), caspase-3 (43.5 versus 13.4%), caspase-8 (44.4 versus 17.1%) and caspase-9 (42.4 versus 10.0%); (ii) the rate of DNA fragmentation was higher (26.3 versus 12.8%); and (iii) sperm ultrastructural examination highlighted a higher percentage of immature (28.0 versus 10.0%) and apoptotic (24.5 versus 18.5%) spermatozoa. FISH study showed predominant normal/balanced spermatozoa (78.34-85.53%). CONCLUSIONS: These results show a predominant proportion of balanced and normal gametes and higher numbers of spermatozoa showing apoptosis and immaturity features in oligoasthenozoospermic Robertsonian translocation carriers than in fertile donors. This suggests defects in spermatogenesis and especially spermiogenesis of these infertile patients.

Predictive value of sperm-FISH analysis on the outcome of preimplantation genetic diagnosis (PGD) for a pericentric inversion inv5(p15.3q11.2) carrier.

BACKGROUND: Pericentric inversions (PIs) are structural chromosomal abnormalities, potentially associated with infertility or multiple miscarriages. More rarely, at meiosis, odd numbers of genetic recombinations within the inversion loop produce recombinant gametes which may lead to aneusomy of recombination in the offspring. METHODS: We report a FISH segregation analysis of an inv5(p15.3q11.2) carrier, both in sperm and blastomeres. In sperm, we directly evaluated the proportion of recombinant gametes and compared the results with chromosomal abnormalities found in blastomeres collected from embryos obtained following a preimplantation genetic diagnosis (PGD) procedure. RESULTS: A total of 7006 sperm nuclei were analyzed. The size of the inverted segment represented 27% of the total length of chromosome 5. The frequencies of balanced chromosomes (normal or inverted), recombinant chromosomes and unbalanced combinations were 97.1, 0.17 and 2.73%, respectively. Of six embryos, PGD FISH analysis revealed that one was a balanced embryo, whereas five were unbalanced and there were no recombinants. CONCLUSIONS: This study demonstrated the value of sperm-FISH analysis in providing reproductive genetic counseling for PI carriers. Our study also highlights the clinical relevance of performing PGD instead of prenatal diagnosis.

Combined FISH and PRINS sperm analysis of complex chromosome rearrangement t(1;19;13): an approach facilitating PGD.

Complex chromosome rearrangements (CCRs) are structural aberrations involving three or more breakpoints on two or more chromosomes. These CCRs result in a high rate of chromosome imbalances potentially leading to subfertility and congenital abnormality. In this study, we analysed meiotic segregation in the sperm of a patient with a familial CCR 46, XY,t(1;19;13)(p31;q13.2;q31)mat included in an intracytoplasmic sperm injection program because of oligoasthenozoospermia. The rearrangement was first identified using conventional and molecular cytogenetic methods. Primed in situ labelling (PRINS) and fluorescence in situ hybridization (FISH) techniques were then combined allowing the simultaneous use of five fluorochromes on the same sperm preparation, for the segregation analysis and the evaluation of the reproductive options for this patient. Segregation analysis was performed in a total of 1822 sperm nuclei from the translocation carrier. The percentage of unbalanced sperm was 75.9%, including 34.1% from 3:3 segregation, 38.2% from 4:2 segregation, 3.5% from 5:1 segregation and 0.05% from 6:0 segregation. Only 14.8% of sperm nuclei were consistent with a normal or balanced chromosome complement. In conclusion, chromosome segregation analysis combining FISH and PRINS was performed in sperm from a CCR carrier using five fluorochromes. These results advance our understanding of the mechanisms of meiotic segregation, and facilitate the assessment of the usefulness of preimplantation genetic diagnosis procedures in CCR couples.

[Could apoptotic markers help the exploration of male infertility?]. / Les marqueurs apoptotiques ont-ils leur place comme marqueurs potentiels de l'exploration de l'infertilité masculine ?

Apoptosis is a cell death program involved in different steps of spermatogenesis, first at puberty, at the beginning of spermatogenesis, then in adult testicles by controlling normal spermatogenesis. As a result, apoptosis deregulation can affect spermatogenesis. Many studies have provided evidence that apoptosis deregulation in germinal cells resulted in male infertility. In addition, apoptosis detection in ejaculated spermatozoa arouses a growing interest in research as a reliable marker of spermatozoon quality. The aim of this review is to summarize our knowledge on physiological apoptosis during spermatogenesis, and then analyse the possibility of using apoptotic markers as selective markers of spermatozoon quality to optimize the rate of success of in vitro fertilization.

[Benefit of human gamete cytogenetics: results and perspectives]. / Intérêt de la cytogénétique des gamètes humains : résultats et perspectives.

In man, the incidence of reproductive failures is high and chromosomal abnormalities remains the major cause of pregnancy wastage. The advent of molecular cytogenetic techniques and assisted reproduction technology have brought forth new approaches for the chromosomal analysis of human oocytes and spermatozoa. The oocyte analyses have evidenced the high rate of chromosomal abnormalities in women and identified premature separation of sister chromatid as a major mechanism in aneuploidy occurrence. High frequencies of aneuploidy have been found in various groups of women, such as patients over 35 or 38 years old, patients with recurrent implantation failures or recurrent miscarriages. The polar body analysis has confirmed the major contribution of premature separation of sister chromatids in aneuploidies and the effect of maternal ageing on its occurrence. In spermatozoa, the efficient adaptation of in situ chromosomal detection techniques has facilitated the segregation analysis of chromosomal abnormalities. Despite the consensus observed in sperm studies of robertsonnian translocations and inversions, new data are required for accurate estimates of imbalances in various types of structural rearrangements. For infertile patients with normal karyotypes, there is significant increase in aneuploidy frequencies, which can be extremely elevated in some groups of subjects, such as patients with large headed spermatozoa syndrome.

Breakpoint characterization: a new approach for segregation analysis of paracentric inversion in human sperm.

Paracentric inversions (PAI) are structural chromosomal rearrangements generally considered to be harmless. Nevertheless, cases of viable recombinants have been reported, indicating the interest of studying the meiotic behaviour of these chromosomal abnormalities. To date, the few studies reported have been performed using either the human-hamster fertilization system or fluorescence in situ hybridization with centromeric or telomeric DNA probes. In order to improve the assessment of meiotic segregation in PAI, we present a new strategy based on the use of bacterial artificial chromosome (BAC) probes which allow a precise localization of chromosome breakpoints and the identification of all meiotic products in human sperm. Sperm samples from carriers of an inv(5) and an inv(14) were used to test this new high-resolution procedure.