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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.

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 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.

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.

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.

Some epidemiological and experimental data in mouse have suggested that there could exist a relationship between sexual abstinence duration and occurrence of chromosomal disorders. In order to determine whether sexual abstinence exerts an effect on the incidence of chromosomal abnormalities in Human, sperm chromosome complements of 4 men were analyzed after fusion with golden hamster eggs. The period of abstinence ranged from 2 days to 15 days. Seven hundred and seventy five karyotypes were obtained. The overall frequency of abnormalities was 9.4% that is not different from controls. There was no correlation between the length of the sexual abstinence and each type of chromosomal abnormality but an indirect effect of paternal aging cannot be excluded.

The incidence of chromosomal abnormalities at conception has been estimated to be very high about 40%; these estimations have been made on indirect evidence provided by karyotyping spontaneous abortions products. Now direct evidence is available, consisting in sperm chromosome analysis by using fertilization of zona-free eggs of the golden hamster. In our experience 175 assays have been performed with modifications of the technique described by Martin et al. (1982) and by Brandriff et al. (1984). Consistent results are obtained since the last 30 assays, using for the first time an R banding technique. Chromosomal analysis of 48 spermatozoa from 7 normal males is reported. The frequency of abnormal sperm complements (21%) is higher than reported by previous reports.

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.

A study of 100 sperm karyotypes from 8 normal subjects was carried out using the technique of in vitro hetero-specific human-hamster fertilisation. 19 of those showed some abnormality. 17% of those were hyperploidies or hypoploidies and 2% showed structural abnormalities. A study carried out at the same time on 34 hamster egg karyotypes showed 6 hypoploidies and 1 hyperploidy. In effect oa cytogenetic technique which is very similar to this one makes it possible to analyse the human oocyte karyotype. The results found in these samples of motile sperms show a higher incidence of abnormality than in other published series, but these figures can not be extrapolated to the composition of in vivo whole sperms.

A cytogenetic analysis of seventy human sperms originating from seven normal subjects is described. Results are compared to those of previously reported studies. The share of male and female contributions in the genesis of chromosomal abnormalities in human zygotes is discussed.

Sperm cytogenetics was carried out using technique of in vitro heterospecific human-hamster fertilization. Sperm of 4 men heterozygous from 4 reciprocal translocations (t(4; 17), t(5; 13), t(6; 7) and t(9; 18] was studied. Segregations were various but a majority of unbalanced complements resulting from adjacent 1 segregations was observed. This prevalence was stronger when the pachytene diagram predisposed the translocation to this mode of imbalance at term.

Using the technique of in vitro human-hamster fertilization, sperm of four men heterozygous for 4 reciprocal translocations--t(4;17),t(5;13),t(6;7), and t(9;18)--was studied. Frequencies of numerical abnormalities unrelated to the translocations range from 8.3% to 13.3%, and the incidence of imbalances ranges from 23.0% to 66.0%. Results are pooled with data from the nine other reciprocal translocations reported elsewhere, and the combined data demonstrate that male meiotic segregation is not random: whatever the type of translocation may be, the distribution of imbalances in sperm is constant, with approximately 72.0% adjacent 1, 18.5% adjacent 2, and 9.5% 3:1 segregations. The same prevalence of adjacent 1 segregations as that reported at term for translocations of paternal origin is observed. There is a strong postzygotic elimination process; for a given translocation it affects selectively the maximum-imbalance zygotes so that imbalanced segregations observed at term are always predetermined.