Biomedical subjects

J Benet

Publications and source records attributed to J Benet.

At least 19 recordsLinked to original sources

PubMed2006

A human tetraploid pachytene spermatocyte as the possible origin of diploid sperm: a case report.

Diploid spermatozoa represent 0.2-0.3% of all spermatozoa in the normal population and cause 8.3% of diandric triploids. Errors in meiosis I and II are the most common mechanisms by which diploid spermatozoa are produced. Endoreduplication before meiosis has been suggested as a possible origin for tetraploid meiocytes, which might, in turn, produce diploid sperm. Synaptonemal complex (SC) spreads of a fertile man were immunolabelled (SCP3, MLH1 and CENP) and hybridized with subtelomere-specific multiplex fluorescent in situ hybridization (stM-FISH) assay for SCs identification. The unexpected finding of a tetraploid pachytene cell and the identification of all of its SCs demonstrate that synapsis and crossover events can occur in human tetraploid cells. Moreover, it indicates that diploid sperm may also originate from mitotic errors (endoreduplication) occurring before meiosis.

Aneuploidy↗

PubMed2006

Behaviour of human heterochromatic regions during the synapsis of homologous chromosomes.

BACKGROUND: Alterations of synapsis can disturb or arrest meiosis and result in infertility. Synaptic abnormalities are frequently observed in infertile patients but also in fertile men. METHODS: The subtelomere-specific multiplex fluorescence in-situ hybridization (stM-FISH) has been applied in combination with immunofluorescence to identify all synaptonemal complexes (SCs) and to analyse those presenting synaptic anomalies in fertile and infertile men. RESULTS: SCs with heterochromatin blocks other than centromere (noncentromeric heterochromatin) presented a higher frequency of gaps (SC discontinuities) and splits (unsynapsed SC regions) at pachytene, the incidences for 9qh, 1qh, 15p and 21p being the highest ones. Inter-individual variability in the incidence of synaptic anomalies in these regions has been observed. In addition, synaptic anomalies in other SC regions are more frequent in infertile cases than in controls. Clear association of the SC15 and SC21 to the XY pair has been seen. CONCLUSION: Noncentromeric heterochromatic regions are the last to synapse. The inter-individual variation observed in the incidence of gaps and splits in these regions may be explained by the heteromorphism of these regions in the general population. The presence of synaptic anomalies in other SC regions may indicate nuclei with a severely affected synapsis. Noncentromeric heterochromatic regions might play a role in the association of autosomal SC15 and SC21 with the XY pair.

Centromere↗

PubMed2006

Crossover frequency and synaptonemal complex length: their variability and effects on human male meiosis.

In this study, immunocytogenetics has been used in combination with the subtelomere-specific multiplex-fluorescent in-situ hybridization (stM-FISH) assay to identify 4681 autosomal synaptonemal complexes (SCs) of two fertile men. Comparisons of crossover maps for each individual SC between two men with extremely different meiotic crossover frequencies show that a low crossover frequency results in (i) a higher frequency of XY pairs and of small SCs without MLH1 foci and (ii) lower frequency of crossovers in the proximity of centromeres. In both cases, the bivalents which most frequently lacked MLH1 foci were the XY pair and the SC21. Analysis of SC length showed that SC arms can be longer or shorter than the corresponding mitotic one. Moreover, for a given SC, the variation in length found in one arm was independent of the variation observed in the other one (e.g. SC1p arms are longer than SC1q arms). The results confirmed that reduction in the crossover frequency may increase the risk of achiasmate small bivalents and that interindividual differences in crossover frequency could explain the variability in the frequencies of aneuploidy in human sperm. How MLH1 foci are positioned within the SC is discussed based on detailed MLH1 foci distributions and interfoci distances. Finally, evidence that the variation of the SC arm length may reflect the abundance of open and of compact chromatin fibers in the arm is shown.

Chromosome Mapping↗

PubMed2006

Studying meiosis: a review of FISH and M-FISH techniques used in the analysis of meiotic processes in humans.

It is well known that chromosome in situ hybridization allows the unequivocal identification of targeted human somatic chromosomes. Different fluorescent in situ hybridization (FISH) techniques have been developed throughout the years and, following the mitotic studies, meiotic analyses have been performed using these different techniques. The introduction of M-FISH techniques to the analysis of meiotic cells has allowed the study of meiotic processes for every individual human chromosome. In this paper, we review the different FISH and M-FISH techniques that have been used on human meiotic cells in both men and women.

Diploidy↗

PubMed2005

Karyotyping of human oocytes by cenM-FISH, a new 24-colour centromere-specific technique.

BACKGROUND: Metaphase II (MII) chromosome complements are difficult to karyotype. The objective of this study was to investigate the efficiency and limitations of centromere-specific multiplex fluorescence in situ hybridization (cenM-FISH), a new 24 colour FISH technique using centromere-specific probes, to analyse the whole chromosome complement within human oocytes. METHODS: Oocytes were donated by 34 patients undergoing ovarian stimulation and IVF. The MII oocytes were analysed by means of cenM-FISH, while the confirmation of results was performed by FISH and/or by analysing the corresponding first polar bodies using comparative genomic hybridization (CGH). RESULTS: A total of 30 cells, corresponding to 16 oocytes and 14 first polar bodies, were successfully karyotyped by either cenM-FISH or CGH. The incidence of aneuploidy was 25%, and eight out of nine aneuploidy events were confirmed by CGH and FISH. CONCLUSIONS: We demonstrate here for the first time that the identification of any numerical abnormality in oocytes is feasible using cenM-FISH. Despite the fact that the fixation efficiency remains low, the present results confirm the advantage of analysing the whole set of chromosomes to make an accurate estimation of the aneuploidy rate in human oocytes.

Adult↗

PubMed2005

Aneuploidy 12 in a Robertsonian (13;14) carrier: Case report.

In translocation carriers, the presence of aneuploidy for the chromosomes unrelated to the rearrangement may lead to an additional risk of abnormal pregnancy or implantation failure. Consequently, it may be important to analyse not only the chromosomes involved in the rearrangement but also the rest of chromosomes. We combined spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to karyotype one unfertilized oocyte and its first polar body (1PB) from a Robertsonian translocation carrier t(13;14) aged 29 years who was undergoing IVF and preimplantation genetic diagnosis (PGD) for translocations and aneuploidy screening. Two out of four embryos were aneuploid, as a result of an adjacent segregation. The unfertilized oocyte had a normal/ balanced constitution of the chromosomes involved in the reorganization. However, this 1PB-metaphase II doublet was aneuploid for chromosome 12, the oocyte being hyperhaploid (24, X, +12) and its 1PB hypohaploid (22, X, -12). The application of CGH for the study of Robertsonian translocations of maternal origin will be useful to study imbalances of the chromosomes involved in the rearrangement, as well as alterations in the copy number of any other chromosome. The combination of PGD for translocations with aneuploidy screening could help to reduce the replacement of chromosomally abnormal embryos.

Abortion, Habitual↗

PubMed2005

Meiotic studies in two human reciprocal translocations and their association with spermatogenic failure.

BACKGROUND: Reciprocal translocations are often associated with infertility in male carriers. However, some carriers present normal semen profiles and are identified because of repetitive pregnancy failures. METHODS: Here, we report two different cases of reciprocal translocations. The first patient carried a t(10;14) and was normozoospermic. The second patient carried a t(13;20) and was azoospermic. Synaptonemal complexes from both carriers were analysed using immunocytogenetic techniques and multi-centromere fluorescent in situ hybridization (cenM-FISH). RESULTS: Associations between the quadrivalent and the sex body or other autosomes were seen only in the t(13;20) carrier. Heterosynapsis was observed only in the t(10;14) carrier. Synaptic pairing abnormalities were seen in 71% of the spreads in the t(13;20) carrier and 30% of the spreads in the t(10;14) carrier. Recombination frequency was decreased in the t(13;20) carrier, but not in the t(10;14) carrier. CONCLUSIONS: By comparing these two different translocation carriers with different fertility outcomes, we discuss the possible mechanisms by which translocations might cause the spermatogenesis process to fail.

Adult↗

PubMed2005

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.

Adult↗

PubMed2005

Segregation of chromosomes in sperm of reciprocal translocation carriers: a review.

Reciprocal translocations, the most frequent structural aberration in humans, are mainly transmitted by one of the parents. In order to analyze the chromosomal content of the spermatozoa from carriers of chromosomal reorganizations, two methods have been used, karyotyping of sperm chromosomes by the human-hamster system and fluorescence in situ hybridization (FISH) in decondensed sperm nuclei. In this work, we review 92 sperm chromosome segregation studies from 85 different reciprocal translocation carriers, including a triple translocation carrier. Using the human-hamster method, a total of 5,818 spermatozoa from 44 reciprocal translocation carriers have been analyzed, 43 of them carrying a single reciprocal translocation and one was a carrier of a double reciprocal translocation. A segregation analysis in a carrier of a t(2;22;11) has been also reported. Carrying out FISH in sperm nuclei, a total of 237,042 spermatozoa from 46 reciprocal translocation carriers have been analyzed. Six of these were also analyzed by the human-hamster system. Taking into account both methods, a total of 76 different reciprocal translocations have been studied. In 74 of these 76 translocations, the reorganization occurs between autosomes, and in the other two, the Y chromosome is involved. Although along general lines, there are similarities between the results obtained by the two methods of analysis, variations are observed when the distribution of the different types of segregations that produce imbalances is compared. As a general rule reciprocal translocation carriers produce more unbalanced sperm than normal or balanced sperm. The results reported also corroborate that the proportion of unbalanced forms depends on the characteristics of the reorganization and that it varies widely. Thus the importance of performing a detailed meiotic behavior analysis for each particular translocation in order to obtain enough information to give adequate genetic counseling is stressed. Aspects as to the possible overestimation of 3:1 segregations or the presence of interchromosomal effects still need to be elucidated.

Chromosome Segregation↗

PubMed2005

Meiotic abnormalities in infertile males.

Meiotic anomalies, as reviewed here, are synaptic chromosome abnormalities, limited to germ cells that cannot be detected through the study of the karyotype. Although the importance of synaptic errors has been underestimated for many years, their presence is related to many cases of human male infertility. Synaptic anomalies can be studied by immunostaining of synaptonemal complexes (SCs), but in this case their frequency is probably underestimated due to the phenomenon of synaptic adjustment. They can also be studied in classic meiotic preparations, which, from a clinical point of view, is still the best approach, especially if multiplex fluorescence in situ hybridization is at hand to solve difficult cases. Sperm chromosome FISH studies also provide indirect evidence of their presence. Synaptic anomalies can affect the rate of recombination of all bivalents, produce achiasmate small univalents, partially achiasmate medium-sized or large bivalents, or affect all bivalents in the cell. The frequency is variable, interindividually and intraindividually. The baseline incidence of synaptic anomalies is 6-8%, which may be increased to 17.6% in males with a severe oligozoospermia, and to 27% in normozoospermic males with one or more previous IVF failures. The clinical consequences are the production of abnormal spermatozoa that will produce a higher number of chromosomally abnormal embryos. The indications for a meiotic study in testicular biopsy are provided.

Chromosome Aberrations↗

PubMed2004

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis.

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.

Adult↗

PubMed2004

From spermatocytes to sperm: meiotic behaviour of human male reciprocal translocations.

BACKGROUND: Human male translocation carriers may present alterations in the meiotic process due to the presence of the translocated chromosomes. The aim of this work was to study the mechanisms that affect meiotic segregation in translocation carriers by analysing different stages of the meiotic process. METHODS: Meiotic studies using fluorescence in-situ hybridization on both spermatocytes and sperm nuclei were performed in two translocation carriers, t(11;17)(q13.1;p11.2) and t(10;14)(q24;q32). RESULTS: A ring configuration was the main type of quadrivalent found in metaphase I. Overall chiasma frequency was significantly decreased in the t(11;17) carrier. In the t(10;14) carrier, chiasma frequency within the interstitial region of chromosomes 10 and 14 was increased and the recombination pattern was modified. As expected from the frequencies of interstitial chiasmata found in metaphase I in the two subjects, the incidence of asymmetric dyads was sporadic in t(11;17) and very high in t(10;14). In both carriers, segregation frequencies observed at metaphase II were not different from the segregation data obtained in decondensed sperm nuclei. CONCLUSIONS: The concordance observed among results obtained in different spermatogenic stages indicates an absence of cellular selection based on chromosomal imbalances. Results obtained in the aneuploidy assay have not provided any evidence for an interchromosomal effect.

Adult↗

PubMed2004

FISH characterization of a dicentric Yq (p11.32) isochromosome in an azoospermic male.

The most common structural rearrangements of the Y chromosome result in the production of dicentrics. In this work, we analyze an abnormal Y chromosome, detected as a mosaic in an azoospermic male ascertained for infertility. FISH with seven different DNA probes specific for Y chromosome sequences (Y alpha-satellite, Y alpha-satellite III, non-alpha-satellite centromeric Y, SRY gene, subtelomeric Yp, subtelomeric Yq, and PNA-tel) and CGH analysis were performed. FISH results showed that the abnormal Y chromosome was a dicentric Yq isochromosome and that the breakpoint was distally in band Yp11.32. Lymphocyte chromosomes showed a mosaicism with 46,X,idicY(qter-->p11.32::p11.32-->qter) (51.7%), 46,XY (45.6%), and other cell lines (2.7%). In oral interphase cells, the mosaicism was 46,XidicY (62.8%), 46,XY (25.7%), 45,X (6.6%), and others (4.9%). The possible origin of this dicentric Yq isochromosome is discussed. Finally, we compare differences in mosaicism and phenotype among three reported cases with the breakpoint at Yp11.32

Adult↗

PubMed2004

The use of a cell-cycle phase-marker may decrease the percentage of errors when using FISH in PGD.

Fluorescent DNA probes are used to characterise the chromosome constitution of preimplantation embryos. FISH is used to select normal or balanced embryos in carriers of balanced chromosomal rearrangements, for embryo sexing or for aneuploidy screening in women of advanced age, who have had recurrent abortions or IVF failures. In most cases, FISH is performed on interphase blastomeres which are asynchronously dividing cells, that can be in G1, S or G2. However, a correct interpretation of a double FISH signal, which may correspond to a split signal, to a replicated chromosome region or to the presence of an extra chromosome is essential to establish an accurate diagnosis. To determine if the cell stage could influence the interpretation of FISH results, we compared the signal characteristics of one locus-specific probe, two different subtelomere region probes, and a centromere region probe in non-dividing Sertoli cells and in proliferating lymphocytes. Most cells had two signals per chromosome pair (i.e., a situation corresponding to G0 in Sertoli cells and to G1 or to a prereplication stage in lymphocytes). Nevertheless, in proliferating cells the percentage of nuclei with a number of signals different from the expected (two unreplicated chromosomes per pair) was different from that found in non-dividing cells (P < 0.05). It was estimated that 10.8% of double dots in dividing cells resulted from DNA replication. The sequence of replication was first the locus-specific region, second a telomere region, and third the centromere. In conclusion, the DNA replication process could result in errors of interpretation (misdiagnosis) in 7% of proliferating cells. Thus, the use of a cell cycle phase-specific marker could avoid errors by indicating the cell stage in which the nucleus analysed is found.

Adult↗

PubMed2004

Characterization of all human male synaptonemal complexes by subtelomere multiplex-FISH.

During meiotic prophase I, homologous chromosomes synapse and recombine. Both events are of vital importance for the success of meiosis. When homologous chromosomes synapse, a proteinaceous structure called synaptonemal complex (SC) appears along the pairing axis and meiotic recombination takes place. The existence of immunolabeling techniques for SC proteins (SCP1, SCP2 and SCP3) and for DNA mismatch repair proteins present in late recombination nodules (MLH1) allow analyses of both synapsis and meiotic recombination in the gametocyte I. In situ hybridization methods can be applied afterwards because chromatin is preserved during cell fixation for immunoanalysis. The combination of both methodologies allows the analysis of synapsis and the creation of recombination maps for each bivalent. In this work we apply the seven-fluorochrome subtelomere-specific multiplex FISH assay (stM-FISH) to human male meiotic cells previously labeled by immunofluorescence (SCP1, SCP3, MLH1, CENP) to assess its utility for human SC karyotyping. This FISH method consists of microdissected subtelomeric probes labeled combinatorially with seven different fluorochromes. Results prove its usefulness for the identification of all human SCs. Furthermore, by labeling subtelomeric regions this one-single-step method enables the characterization of interstitial and terminal SC fragments and SC delineation even if superposition is present in pachytene spreads.

Cytogenetic Analysis↗

PubMed2003

Multiple aneuploidies in the oocytes of balanced translocation carriers: a preimplantation genetic diagnosis study using first polar body.

Preimplantation genetic diagnosis (PGD) of first polar bodies (1PBs) has been used in carriers of balanced chromosomal reorganizations and also for aneuploidy screening. Although an acceptable number of normal or balanced embryos is usually obtained using PGD in translocation carriers, the pregnancy rate is disappointingly low. To determine whether aneuploidy of chromosomes not involved in the chromosome rearrangements could be the cause of the low pregnancy rates achieved, the present authors analysed the segregation products of three translocation carriers, t(8;13)(q24.1;q22) and two Robertsonian (Rob)(13;14), using 1PBs, and afterwards another eight chromosomes in the same 1PBs, for a total of 10 chromosomes in each 1PB, that is chromosomes 1, 8, 13, 14, 15, 16, 17, 18, 21, 22 and X. In the reciprocal translocation, chromosomes with different chromatids due to meiotic recombination were found. Only one out of nine 1PBs was normal for the reorganization products but no aneuploidies were found after PGD in this case. In the two balanced Rob(13;14), six out of 12 and four out of 11 1PBs were normal or balanced for the reorganization but only one oocyte was euploid for all the chromosomes analysed in each case; a single embryo transfer was made in both but no pregnancy was achieved. The incidence of aneuploidy for the chromosomes not involved in the Robertsonian translocations was extremely high (91.7% and 81.8%). Extra chromosomes were present in most of the aneuploid oocytes (81.8% and 90%). The reason for this increase could be the tendency to non-disjunction related to advanced maternal age combined with an interchromosomal effect resulting in the presence of synaptic errors in other chromosome pairs.

Adult↗

PubMed2002

Aneuploid and unbalanced sperm in two translocation carriers: evaluation of the genetic risk.

Translocation carriers have an increased risk of reproductive failure or affected offspring, because of the production of unbalanced gametes by meiotic segregation or the possible presence of interchromosomal effects (ICE). We therefore performed an analysis of meiotic segregation using the human-hamster IVF technique, and an aneuploidy assay for chromosomes 6, 18, 21, X and Y, using dual and triple-colour fluorescence in-situ hybridization, in two translocation carriers, t(1;13)(q41;q22) and t(3;19)(p21;p13.3). Sperm chromosome complements were analysed by whole chromosome painting. The frequencies observed for alternate, adjacent I, adjacent II and 3:1 segregations were, for t(1;13), 41.6, 41.6, 14.5 and 2.3% respectively, and for t(3;19), the frequencies were 39.1, 35.9, 21.8 and 3.2% respectively. More than 20,000 sperm per subject were analysed in the aneuploidy assay. Disomy 21 was found to be higher than other autosome disomies. Evidence for a possible ICE was found only in t(3;19). This study has shown that unbalanced sperm are more frequent than aneuploid sperm in the total sperm population. However, data in the literature suggest that the importance of each aberrant population seems to be more significant for embryo viability than would be expected from the increases in the percentages of abnormal sperm.

Aneuploidy↗

PubMed2001

Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei.

Meiotic segregation of a t(4;8)(q28;p23) translocation carrier was determined by two different methods to compare the final results. A total of 352 sperm chromosome complements, obtained after human-hamster in vitro fertilisation, were analysed by whole chromosome painting, and 6590 sperm heads were studied by fluorescence in situ hybridisation (FISH). Frequencies of alternate, adjacent I, adjacent II and 3 : 1 segregations were, for sperm chromosomes, 35.5, 33.2, 19.9 and 11.3% respectively. For sperm head analysis, results were 30.5, 28.5, 20.5 and 19.5% respectively. There were no statistically significant differences between the two methods with respect to the observed frequencies of sperm with balanced and unbalanced chromosome constitutions. Among unbalanced gametes, the methods differed only in the frequency of 3 : 1 segregation (chi(2), P<0.0001). Different factors that could explain this result are discussed. To determine possible interchromosomal effects, multicolour FISH was used on sperm heads. Disomy rates of sex and 18 chromosomes were higher in the translocation carrier than in the control. The differences observed were statistically significant (P<0.0001 for chromosomes X and 18, and P=0.0091 for chromosome Y).

Adult↗