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pachytene stage to round spermatids. Furthermore, it has been reported that sex-chromosome univalency is relatively common in human MI spermatocytes (from infertile males).10 The pairing features of these chromosomes (restricted to the short pseudoautosomal regions) promote the presence of univalents and explain the high incidence of sex-chromosome aneuploidies in human spermatozoa from infertile patients.11 Nevertheless, the influence of univalency in the territorial organization of human spermatocytes has not been ascertained. In this study, we have determined the effect of the presence of sex-chromosome univalents on the territorial organization of MI human spermatocytes from infertile patients with a normal somatic karyotype (46,XY). Chromosome proximity analysis was performed in a total of 1944 chromosome units from 81 MI,24,X,Y(MI bearing the X and Y chromosomes as univalents). MI spermatocytes were obtained from testicular biopsies from twenty-six 46,XY infertile men (range 1–10 MI per individual). Biopsies were obtained for diagnostic purposes to evaluate the incidence of meiotic chromosomal abnormalities in spermatocytes at MI,10 and surplus material was used for this study. The study was approved by the Institutional Review Board of the collaborating centers (Centre de Reproducció Assistida Fecunmed, Fundació Puigvert, Institut Marquès, Institut Universitari Dexeus and Instituto de Reproducción CEFER). In every instance, all of the patients signed their informed consent about participation in the study and the centers provided all data while preserving patient anonymity. Testicular biopsies were mechanically disaggregated, and cell suspensions were fixed and dropped onto dry slides.10 Chromosome preparations were processed following a protocol that combine Leishman staining (PanReac AppliChem, Castellar del Vallès, Spain) and multiplex fluorescence in situ hybridization (M-FISH, Spectra VysionTM Assay Protocol, Vysis Inc., Downers Grove, IL, USA) procedures (Figure 1).10 Briefly, MI images from Leishman-stained slides were captured and coordinates were noted. Slides were further processed for M-FISH analysis, which enabled unequivocal chromosome identification based on a distinctive color pattern. MI cells were re-located and re-analyzed after the FISH procedure. Proximity analysis was performed as it has been previously described.2 Briefly, in each cell, nearby chromosome units (including bivalents and univalents) were determined for each unit contrasting Dear Editor, In interphase nuclei, chromosomes occupy specific localizations called chromosome territories (CTs). Distribution of CTs has been related to chromosome size and gene density and is known to influence gene expression.1 This pattern of organization appears to be a widespread nuclear feature, both in somatic and germ cells. In spermatogenic cells, we have previously revealed a somatic-like distribution of bivalents at metaphase I (MI) with a nonrandom bivalent disposition and a preferential association between bivalents according to chromosome size, morphology, and gene density.2 Other studies have revealed preferential proximity of chromosomes 15 and 22 to the sex-chromosomes bivalent in MI.3 Moreover, a nonrandom chromosome distribution has also been reported in sperm nuclei.4 Although the functionality of CTs in male germ cells is not fully understood, it has been proposed that it has a role in the regulation of gene expression during spermatogenesis and early embryo development.5 Accordingly, alterations in CTs in germ cells could have implications for genome regulation leading to male infertility. Our research group has provided the first evidence of alteration in CTs in the presence of Robertsonian translocations (rob) in MI human spermatocytes.6 The impaired fertility in rob carriers is mainly due to the production of chromosomally abnormal spermatozoa because of the regular segregation of the chromosomes involved in the reorganization and the occurrence of interchromosomal effects.7 However, we have also revealed in rob carriers that, the trivalent configuration of the reorganized chromosomes disturbs the overall chromosome positioning at MI.6 Thus, the disruption of MI CTs could be an additional contributory factor to infertility associated with this kind of chromosomal reorganization.6 Interestingly, in human spermatogenesis, the XY chromosomes organize in the sex vesicle at prophase I,8 and in the postmeiotic sex chromatin (PMSC) body in spermatids.9 In terms of gene expression and CTs organization, sex chromosomes configure a silent compartment where chromosomes appear inactivated from the LETTER TO THE EDITOR

Unpaired sex chromosomes in metaphase I human spermatocytes locally modify autosomal bivalents positioning