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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.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.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.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.

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.

Resume Les anomalies chromosomiques sont a l'origine de la majeure partie des pertes embryonnaires en periode pre- et postimplantatoire. Pour l'essentiel, elles sont issues d'erreurs de segregation chromosomique au cours de la meiose feminine, et en particulier lors de la premiere division meiotique. En consequence, l'analyse chromosomique des ovocytes matures est une source d'informations essentielles pour l'etude de la formation et de l'etiologie des anomalies chromosomiques dans l'espece humaine. Durant les 40 dernieres annees, ce domaine d'investigation n'a jamais cesse de progresser au gre des innovations technologiques. Ainsi, des etudes cytogenetiques conventionnelles et des etudes de cytogenetique moleculaire ont ete menees et ont fourni de nombreuses donnees sur l'incidence et la distribution des anomalies chromosomiques dans les gametes femelles. Toutefois, les resultats rapportes presentent d'importantes variations qui traduisent les limites inherentes a l'application de ces techniques sur les ovocytes humains. Les donnees les plus fiables rapportent des taux d'anomalies chromosomiques de 15 a 20 % dans les ovocytes humains et ont mis en evidence l'implication conjointe des mecanismes de non-disjonction chromosomique et de separation des chromatides sœurs dans la survenue des aneuploidies ovocytaires. Par ailleurs, les etudes les plus recentes fondees sur de larges echantillons d'ovocytes ou de globules polaires ont permis de confirmer l'effet de l'âge maternel sur l'incidence des anomalies chromosomiques.

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.

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.

Both PRimed IN Situ (PRINS) and Peptide Nucleic Acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing.

Chromosomal abnormalities account for the majority of pre- and post- implantation embryo wastage in humans. Most of these abnormalities result from maternal meiotic errors, which preferentially occur during the first meiotic division. Consequently, the cytogenetic analysis of human oocytes has then been considered as a highly valuable source of data for the investigation of both the occurrence and the origin of chromosomal abnormalities in human. During the last 4 decades, the cytogenetic analysis of human oocytes has never stopped progressing, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome non-disjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidy in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies.

BACKGROUND: The comparison of male and female meiotic segregation patterns for individuals carrying identical reciprocal translocations has been rarely reported in mammalian species. The main comparative study involving males and females with comparable genetic background has been performed in the mouse. Swine is another relevant animal model species for meiotic studies. Here we present the segregation patterns determined for sows carrying one of the two following reciprocal translocations: 38, XX, rcp(3;15)(q27;q13), and 38, XX, rcp(12;14)(q13;q21). These segregation data were compared to those previously obtained for closely related boars carrying the same balanced chromosomal rearrangements. METHODS: Dual colour in situ hybridization of whole chromosome painting probes was carried out on metaphases of in vitro-matured oocytes II. Segregation results were obtained for 118 and 206 metaphases II respectively for the two translocations. RESULTS: Significant differences between sexes were demonstrated for both rearrangements. For instance, for the 3/15 translocation, the chromosomally unbalanced gametes were of different origin: preponderance of the adjacent-I segregation in the male (31.4%), and of the adjacent-II (14.3%) and 3:1 (14.3%) segregations in females. For the 12/14 translocation, the proportion of balanced gametes was greater in males than in females (75.9 and 59.4% respectively). CONCLUSION: This study is a new scientific contribution to compare the segregation patterns of male and female carriers of identical chromosomal rearrangements. The results obtained are consistent with those previously reported in mice. Hypotheses to interpret the observed differences between the two translocations, as well as between the male and female segregation patterns, are formulated and discussed.

BACKGROUND: The t(13;22) Robertsonian translocation constitutes a rare form of rearrangement between acrocentric human chromosomes. Most of the meiotic segregation studies of human Robertsonian translocations have been performed on common t(13;14) and t(14;21) translocations. Analysis of the chromosomal constitution in sperm of Robertsonian translocation carriers is of great interest for assessing the risk of unbalanced forms and adapting genetic counselling. In the present study, we present the first meiotic segregation study of a t(13;22) Robertsonian translocation in human sperm. METHODS: A total of 11 787 sperm nuclei were scored using two distinct FISH labelling techniques, i.e. the locus-specific probes (LSI) method and the whole chromosome painting (WCP) technique. RESULTS: The frequency of normal or balanced sperm resulting from alternate meiotic segregation was 86%. Incidences of unbalanced complements resulting from adjacent segregation modes were 12.79% and 14.36% in LSI and WCP assays, respectively. No significant excess of nullisomy or disomy for the affected chromosomes was observed. CONCLUSIONS: Similar results in segregation were obtained with the two techniques, demonstrating the efficiency of the two strategies for the direct segregation analysis of Roberstsonian translocations. The results obtained indicated a moderate meiotic production of imbalance. This study shows that the rare Robertsonian translocation (13;22) displays a similar distribution of balanced and unbalanced sperm patterns as the common Robertsonian translocations previously studied. This suggests that the behaviour of acrocentric chromosomes was similar in all cases of centric fusion.

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We report a new multicolor PRINS procedure for chromosome identification on human sperm. Based on the direct in-situ mixing of the colors of the fluorochromes (FITC, TRITC, Cascade Blue) incorporated in sequential PRINS reactions, this method facilitates rapid distinct labeling of 3 or 4 chromosomes. Each PRINS reaction consists of a unique 4 minute step for annealing and elongation. The method was successfully tested on lymphocytes and spermatozoa. Estimates of disomy were performed for chromosomes 7, 9 and 16 on sperm samples from 2 healthy donors. There was no significant difference between the disomy rates obtained with the conventional two-color PRINS technique and this new three-color procedure. By simplifying the multicolor PRINS protocol, this new protocol should facilitate the use and adaptation of PRINS to various cytogenetic applications.

Chromosomal races of the house mouse (Mus musculus domesticus) bear Robertsonian (Rb) fusions, which consist of centric translocations between two non-homologous acrocentric chromosomes. The high level of diversity of these fusions in house mice is generated by de-novo formation of Rb fusions and subsequent whole-arm reciprocal exchanges (WARTs). This paper describes the spontaneous occurrence of a new Rb fusion, Rb(4.19), in progeny of wild-derived house mice segregating for Rb(4.12). The chromosomal mutation was traced to a female which exhibited germline and somatic mosaicism indicating an early embryonic origin of the mutation. FISH analysis of centromerically-located ribosomal genes suggested that no modification was observed on chromosomes 12 and 19 prior to or following the occurrence of Rb(4.19). Distribution of telomeric sequences showed that both Rb fusions lacked telomeres in their centromeric regions. It is argued that this spontaneous mutation most likely originated by single whole-arm reciprocal translocation (WART) between Rb(4.12) and an acrocentric chromosome 19, resulting in Rb(4.19) and a neo-acrocentric chromosome 12. Sequences required for centromeric function and proximal telomeres would have been transferred to the neo-chromosome 12 from chromosome 19 during the translocation. The existence of such WARTs which generate derived acrocentric chromosomes has several implications for chromosomal evolution in house mice.

Both detection and chromosomal analysis of fetal cells present in the maternal circulation can be performed using Histopaque double density gradient centrifugation followed by primed in situ (PRINS) labeling technique. This approach has been tested on blood samples from 15 pregnant women and 6 control donors with primers specific for chromosomes 9, X and Y. The cell separation technique allows recovery of both mononuclear cells and polynuclear cells with a 97% efficiency. PRINS labeling was successful in 100% cells from control blood samples. Among patient samples, 2 "false-negative" results were observed. These preliminary results suggest that the present protocol might be efficient for non-invasive prenatal chromosome analysis.

The primed in situ (PRINS) labeling technique has been used for the interphase cytogenetic investigation of 3 colon cancer cell lines (Caco-2, TC7 and PF1 1) derived from a same primary tumor. A panel of 10 chromosome-specific primers (for chromosomes 1, 2, 3, 7, 8, 9, 10, 13, 16 and 18) has been utilized in simple and double color PRINS reactions. Each cell line displayed a heterogeneous distribution of copy number for several chromosomes. The karyotypic heterogeneity was also significant between the 3 cell lines. These data indicate the common occurrence of chromosome heterogeneity in tumoral cell lines and demonstrate the feasibility of interphase PRINS procedure for analysis of numerical changes in tumoral cells.

The primed in situ (PRINS) labeling technique was developed as an alternative method to classical cytogenetics and in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed using Alu specific primers generating the painting of all human material associated with R like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Due to the use of synthetic oligonucleotide primers and of directly labeled haptens. PRINS method avoid repetitive probes preparation, eliminates secondary amplification of signals and the whole process can be performed within a timespan of 1 hour. Providing qualitative and quantitative answers, the simple PRINS method appears very well adapted to the specific problematic of somatic hybrids as for their characterization than for their periodic controls imposed by their instability. The method has been tested on 4 human-rodent hybrid cell lines. In particular, the somatic hybrid clone ALE 4 was shown to be monochromosomal for the der(11) from the reciprocal translocation t(11:22).

We propose a simple, fast and inexpensive method of identification of human centromeres on metaphasic chromosomes and interphasic nuclei. This is based on in situ hybridization of labelled oligonucleotides. The efficiency of the methodology was demonstrated on cytogenetic preparations from human heteroploid and human x hamster hybrid cell lines and also on frozen tissue sections using an oligonucleotide specific for the alpha-satellite DNA of chromosome 1. Three versions of this oligonucleotide respectively labelled with 1, 4 and 10 fluorescein molecules were synthesized. The signal intensity provided by the oligonucleotide coupled with 4 fluoresceins allowed unambiguously the detection of the chromosome and the establishment of its ploidy using a classical cytogenetic microscope without the need for an amplification procedure. The use of different fluorochromes and possibly combination with an unlabelled elongation in 3' of the oligonucleotides which stabilize its hybridization, lead to a simple multicolour method. Preliminary quantification of the signals obtained by in situ hybridization of labelled oligonucleotides and comparison with those obtained by primed in situ labelling (PRINS) using the same nucleotides as primers, suggest that the elongation generated by PRINS may be very short compared with a PCR in solution. This limited efficiency of the in situ elongation may reflect the present difficulties of PRINS and DISC PCR (direct in situ single copy polymerase chain reaction) with primers specific for non-repetitive sequencies.

The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in

Direct in situ labeling of human spermatozoa was performed using the PRINS method. This technique is based on annealing of specific oligonucleotide primers, and subsequent primer extension by a Taq DNA polymerase. The reaction was carried out on a programmable temperature cycler, and labeling was obtained in a 1-hr reaction. The method was successfully tested with specific primers for chromosomes 13, 16, and 21. This suggests that PRINS may be a fast and reliable technique for detecting aneuploidies.

The PRINS method allows a rapid and specific detection of human chromosomes in situ. We have adapted the PRINS protocol to human sperm. Estimates of disomy have thus been performed for chromosomes 9, 12, 16 and 21 by using alpha-satellite DNA specific primers. The frequencies of disomy ranged from 0.27% to 0.31%. No significant difference was found. These data agree with the hypothesis of an equal distribution of non-disjunctions among chromosomes in male meiosis.

In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities.

To evaluate the effects of long abstinence periods on semen characteristics.Semen analysis was performed on six men after various sexual abstinence periods (from 2 to 18 days). The variations in semen parameters were analyzed statistically as a function of the duration of abstinence.The Reproductive Biology Laboratory at the University of Montpellier Medical School.Lengthy sexual abstinence was found to affect all semen characteristics. Semen volume and concentration and total sperm count showed significant increases, whereas motility and normal morphology decreased significantly with duration of abstinence. Significant changes in the percentage of normal sperm forms were observed after more than seven days' abstinence.The study indicates that the influence of long sexual abstinence on semen quality varies with the variable considered. With regard to fertility, a long abstinence period might induce senescence of spermatozoa.

The PRINS technique has been used to rapidly detect chromosomes 21 in both metaphases and interphase nuclei. A specific labeling was obtained in preparations from lymphocytes and amniotic fluid cells. The method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. PRINS is an interesting alternative to in situ hybridization for cytogenetic diagnosis and physical mapping.

A cytogenetic analysis was performed on a sample of 411 human grade IV embryos (i.e. poor morphological quality embryos, never transferred in our in vitro fertilization (IVF) pro Gram) in order to investigate the chromosomal status of these embryos. One hundred eighteen were successfully karyotyped from at least one metaphase. Only 10% displayed normal diploid metaphases. Aneuploidy was the most frequently observed abnormality, with a rate of 36.4%. Six cases of single chromatids were noted and 9 embryos showed structural aberrations. Polyploidy (from 3n to 7n) and haploidy were also observed, suggesting parthenogenetic activation, polyspermy or chromosomal duplication. Mosaicism constituted 6% of the abnormalities. Thirty embryos exhibited fragmented chromosome sets which might result from in vitro delayed fertilization.

Epidemiological and mouse cytogenetic studies have suggested the existence of a positive relationship between the frequency of chromosomal abnormalities and the duration of male sexual abstinence. A cytogenetic analysis of human spermatozoa was performed with 4 men after periods of sexual abstinence ranging from 2 to 15 days. Using hamster eggs for penetration, 934 sperm chromosome complements were obtained. The overall frequency of sperm chromosomal abnormalities was 9.4%. There was no correlation between the length of sexual abstinence and each type of chromosomal abnormality. Our results do not support the hypothesis of an increased risk of aberration with duration of abstinence.

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.