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.

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).

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.

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.

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.

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.

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.

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.

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