Cristina Templado

The north-west coast of Spain was heavily contaminated by the Prestige oil spill, in 2002. Individuals who participated in the clean-up tasks showed increased chromosome damage two years after exposure. Long-term clinical implications of chromosome damage are still unknown. To realize a follow-up genotoxic study to detect whether the chromosome damage persisted six years after exposure to the oil. Follow-up study. Fishermen cooperatives in coastal villages. Local fishermen who were highly exposed (n = 52) and non-exposed (n = 23) to oil seven years after the spill. Chromosome damage in circulating lymphocytes. Chromosome damage in exposed individuals persists six years after oil exposure, with a similar incidence than those previously detected four years before. A surprising increase in chromosome damage in non-exposed individual was found six years after Prestige spill vs. those detected two years after the exposure. The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the approximately 300,000 individuals who participated occasionally in clean-up tasks. The persistence of chromosome damage detected in exposed individuals six years after oil exposure seems to indicate that the cells of the bone marrow are affected. A surprising increase in chromosome damage in non-exposed individuals detected in the follow-up study suggests an indirect exposition of these individuals to some oil compounds or to other toxic agents during the last four years. More long-term studies are needed to confirm the presence of chromosome damage in exposed and non-exposed fishermen due to the association between increased chromosomal damage and increased risk of cancer. Understanding and detecting chromosome damage is important for detecting cancer in its early stages. The present work is the first follow-up cytogenetic study carried out in lymphocytes to determine genotoxic damage evolution between two and six years after oil exposure in same individuals.

In humans, the most common chromosomal abnormality is aneuploidy. Because the majority of aneuploid conceptuses die during the early stages of embryonic development, an accurate estimate of the frequency of aneuploidy at conception can only be assessed by directly studying the gametes. The vast majority of aneuploidies arise de novo as a result of sporadic chromosome missegregation in paternal or maternal meiosis. In this review, we present the basic current knowledge about the incidence of aneuploidy in human spermatozoa in the general population and in patient populations where elevated levels of sperm aneuploidy are observed. These include infertile patients, patients with abnormal somatic karyotypes, and individuals exposed to certain environmental/lifestyle hazards. The clinical impact of increased levels of aneuploidy is discussed. We then focus on the non-disjunction mechanisms that cause aneuploidy during spermatogenesis and the factors that predispose to non-disjunction in male germ cells followed by an analysis of the sex differences in the incidence of gamete aneuploidy. Recent meiotic studies using multiplex-FISH on three fertile men have revealed that the frequency of conservative aneuploidy of metaphase II spermatocytes is similar to that observed in non-inseminated oocytes of young women. These findings suggest that the differences in the incidence of aneuploidy between spermatozoa and oocytes are not due to differences in chromosome segregation errors but rather to more effective checkpoint mechanisms in spermatogenesis than in oogenesis.

In humans, little is known about the mechanisms of non-disjunction working in male meiosis, although considerable attention has been given to these mechanisms in female meiosis. The present study explores the origin of meiotic non-disjunction during human spermatogenesis and the chromosomes most commonly involved in this process. We used Multiplex fluorescence in situ hybridization to carry out meiotic analyses in metaphase I (MI) and metaphase II (MII) spermatocytes from three fertile donors. Testicular biopsy was obtained during a vasectomy procedure. We examined a total of 317 MI and 248 MII spermatocytes. The frequency of numerical chromosome abnormalities at MII (14.5%) was 5.5 times higher than at MI (2.5%). We observed 88 (27.7%) spermatocytes I with chromosome bivalents with a low chiasma count, usually small chromosomes displaying two separated univalents. Chromosomes X, Y and 21 were the most commonly found as achiasmate chromosomes at MI and the most frequently involved in disomy at MII. Hyperploidy frequency in spermatocytes II (disomy) was significantly higher (P< 0.001) than that found in spermatocytes I (trisomy). Achiasmate non-disjunction and premature separation of sister chromatids appear to be the two main non-disjunction mechanisms during the first meiotic division in human spermatogenesis, and both mechanisms contribute equally to the genesis of aneuploidy. The elevated frequencies of disomy detected in spermatocytes II are significantly higher than those previously described in human spermatozoa, suggesting the existence of a postmeiotic checkpoint monitoring numerical abnormalities.

We reviewed the frequency and distribution of disomy in spermatozoa obtained by multicolor-FISH analysis on decondensed sperm nuclei in (a) healthy men, (b) fathers of aneuploid offspring of paternal origin and (c) individuals with Klinefelter syndrome and XYY males. In series of healthy men, disomy per autosome is approximately 0.1% but may range from 0.03 (chromosome 8) to 0.47 (chromosome 22). The great majority of authors find that chromosome 21 (0.18%) and the sex chromosomes (0.27%) have significantly elevated frequencies of disomy although these findings are not universal. The total disomy in FISH studies is 2.26% and the estimated aneuploidy (2× disomy) is 4.5%, more than double that seen in sperm karyotypes (1.8%). Increased disomy levels of low orders of magnitude have been reported in spermatozoa of some normal men (stable variants) and in men who have fathered children with Down, Turner and Klinefelter syndromes. These findings suggest that men with a moderately elevated aneuploidy rate may be at a higher risk of fathering paternally derived aneuploid pregnancies. Among lifestyle factors, smoking, alcohol and caffeine have been studied extensively but the compounding effects of the 3 are difficult to separate because they are common lifestyle behaviors. Increases in sex chromosome abnormalities, some autosomal disomies, and in the number of diploid spermatozoa are general features in 47,XXY and 47,XYY males. Aneuploidy of the sex chromosomes is more frequent than aneuploidy of any of the autosomes not only in normal control individuals, but also in patients with sex chromosome abnormalities and fathers of paternally derived Klinefelter, Turner and Down syndromes.

This study explores the relationship between sperm structural aberrations and age by using a multicolor multichromosome FISH strategy that provides information on the incidence of duplications and deletions on all the autosomes. ToTelvysion kit (Abbott Molecular, Abbott Park, IL, USA) with telomere-specific probes was used. We investigated the sperm of 10 male donors aged from 23 to 74 years old. The donors were divided into two groups according to age, a cohort of five individuals younger than 40 and a cohort of five individuals older than 60 years. The goal of this study was to determine (1) the relationship between donor age and frequency and type of chromosome structural abnormalities and (2) chromosomes more frequently involved in sperm structural aberrations. We found that the older patients had a higher rate of structural abnormalities (6.6%) compared with the younger cohort (4.9%). Although both duplications and deletions were seen more frequently in older men, our findings demonstrate the presence of an excess of duplications versus deletions in both groups at a ratio of 2 to 1. We demonstrate that the distribution of duplications and deletions was not linear along the chromosomes, although a trend toward a higher rate of abnormalities in larger chromosomes was observed. This work is the first study addressing the frequencies of sperm chromosome structural aberrations of all autosomes in a single assay thus making a contribution to the clarification of the amount and origin of damage present in human spermatozoa and in relation to age.

Three new cases of low chiasma frequency in infertile men are described. In the first case, all cells showed abnormal diakineses; in the second, 20% of the diakineses were desynaptic, while the remaining 80% were normal; in the third, all diakineses were desynaptic and showed chromosome fragmentation. The possible mechanisms leading to asynapsis, desynapsis or precocious chiasma terminalization are discussed.

Most meiotic studies in metaphase spermatocytes have been carried out with classic cytogenetic techniques. The aim of this work was to adjust the centromere-specific multicolour fluorescence in situ hybridization (cenM-FISH) procedure to spermatocyte metaphases I and II in order to improve the identification of meiotic chromosome abnormalities. A total of 168 spermatocytes I and 66 spermatocytes II from two fertile males have been studied using cenM-FISH. The mean frequency of meiotic abnormalities (synaptic, numerical and structural errors) found in metaphases I and II was 22.1 and 3.0%, respectively. The cenM-FISH technique has not only enabled the individual identification of chromosomes involved in meiotic disorders, but also increased the number of analysable cells, principally at metaphase II stage. CenM-FISH is a useful tool to study the meiotic chromosomal disorders and mechanisms leading to chromosomally abnormal spermatozoa.

Meiotic disorders result in meiotic arrest and aneuploid spermatozoa, leading to male infertility, spontaneous abortions or affected offspring. We carried out meiotic studies in an infertile male to detect meiotic nondisjunction mechanisms leading to aneuploidy in spermatogenesis. Meiotic studies were performed in testicular and semen samples from a 38-year-old teratozoospermic male with normal somatic karyotype and a history of spontaneous abortions. We analysed 262 spermatocytes (69 pachytene cells, 106 metaphases I (MI), 87 metaphases II (MII)) by multiplex-fluorescence in situ hybridization and 20 193 spermatozoa by multicolour-FISH with probes for chromosomes 9, 10, 15, 21, X and Y. The results indicate high increase of 21 and XY disomies, as well as diploidy in both spermatocytes at MII and spermatozoa (P < 0.0001). Achiasmate segregation of sex chromosomes was found in 3.4% of spermatocytes II, preceded by early-dissociated XY bivalent at MI (41.5% of cells). We also detected premature separation of sister chromatids (PSSC) in 4.6% of MII. This individual presents high levels of numerical abnormalities in germ cells, caused by two different nondisjunction mechanisms during meiosis I. To our knowledge, this work represents the first time that PSSC has been demonstrated in human male germ cells.