Risultati finali
Telomeres and telomerase play key roles in the maintenance of genome stability. Chiara Mondello, in collaboration with Elena Giulotto, has shown that in telomerase expressing cells, very short telomeres do not increase cellular and chromosomal radiosensitivity, as they do in telomerase negative cells. On the other hand, they have shown that in telomerase deficient mouse cells, gene amplification, a typical manifestation of genome instability observed in tumour cells, is not detectable, indicating that telomerase is required for the occurrence of this process. The lack of gene amplification in telomerase deficient cells could be due to the inability of the deficient cells to stabilize amplified chromosomes with new telomeres. Alternatively, telomerase could have an indirect role by controlling the expression of other genes involved in the process. These results highlight the complexity of the role of telomeres and telomerase in the preservation of genome integrity.
Since telomerase is expressed in the vast majority of the tumours, the indication that telomerase is essential for gene amplification suggests that the use of drugs inhibiting telomerase could also decrease the occurrence of gene amplification, thus reducing tumour aggressiveness. These aspects are particularly relevant for the future development of personalized therapeutic protocols.
Human mammary epithelial cells derived from normal breast tissue from three women subjected to spontaneous reduction were analysed. These cells may spontaneously overcome the normal proliferation limits by loosing expression of p16 protein. The karyotype evolution of HMECs was followed by means of an exhaustive cytogenetic analysis. As an overall result, there was an increase in the frequency of aberrations per cell throughout the culture. Anna Genesca's results confirm that the individual chromosome arms carrying the shortest telomeres are the ones involved in fusions. Moreover, the analysis of the specific chromosomes with the shortest telomeres in post-selection HMECs derived from the three women provided evidence that the set of chromosome arms showing preferential eroded telomeres depends on each individual. This variability may be responsible for the observed karyotype differences among tumors of the same type. Most importantly, this partner identified sister chromatid fusion as the first event in generating genomic instability in HMECs. During HMEC growth, double strand breaks are generated by both fused chromosomes as well as individual chromosomes with fused chromatids entering BFB cycles. These broken extremities are able to join other broken ends or eroded telomeres, producing massive instability at the latter passages of the cell culture.
Evidence is accumulating that telomeres may be involved in cellular as well as organismal responses to ionising radiation. Anna Genesca's laboratory analysis identified dysfunctional telomeres joined to radiation-induced DSBs in HMECs, providing the first conclusive evidence of this type of aberration in primary human cells. In addition, telomere-DSB aberrations increased with radiation exposure and are probably responsible for the increased radiation sensitivity of HMECs with short telomeres. From these results, it seems reasonable to conclude that, given a radiation-induced DSB in a chromosome, the probabilities of correct rejoining are dramatically different in cells with normal telomere length in comparison to cells with critically short telomeres.
In cells with normal telomere length, the broken chromosome can only rejoin another broken end, with a high probability of restoring the original chromosome if the number of breaks coexisting in that cell is low. On the contrary, in cells with critically short telomeres, there may be several uncapped chromosomes to which the broken end can join, thus making the restoration of the original chromosome more unlikely. As a consequence, higher radiation sensitivity is observed in HMECs with short telomeres in comparison to normal telomere length HMECs. Provided that it is known that HMECs shorten telomeres with population doublings in vitro (age in vivo) and inactivate expression of p16INK4a both in vitro and in the mammary gland, it might be important to re-evaluate the risks facing women of advancing age when exposed to radiation for diagnostic purposes.
Chromosome fusions are observed with high frequency in cells with defects in components of the standard pathway of DNA double strand break repair which suggests the operation of an alternative repair pathway that is capable of joining together eroded telomeric sequences when the canonical pathway is inactivated. Although DNA ligase III is predominantly considered a component of single strand break and base excision repair, our observations hint to a potential role in DNA DSB rejoining as well. DNA ligase III may be a bona fide component of DNA DSB repair by NHEJ, where it may function either alone or together with XRCC1 and PARP-1. This is in line with the ability of the purified enzyme to ligate plasmid DNA under physiological conditions and is directly supported by recent in vivo and in vitro results. Backup pathways of NHEJ operating independently of DNA ligase IV were also suggested by recent genetic experiments in Drosophila melanogaster and are shown to be involved in the repair of DSBs induced either after exposure to radiation. Evidence is thus mounting that NHEJ pathways operating independently of DNA-PK and DNA ligase IV operate in a variety of organisms.
The results presented allow the inference that these pathways of end joining will contribute significantly to the ability of cells of cope with radiation damage and should therefore contribute to their radioresistance to killing. However, because these pathways are error prone, they contribute to the development of cancer as well as to the fusion of chromosomes, particularly when telomeres erode. George Iliakis' work suggests that protection of chromosome ends from illegitimate end joining is likely mediated by the structure of the telomeres and the binding of specific proteins to telomeric sequences, rather than by an inherent inability of the NHEJ apparatus to deal with telomeric ends. Since end joining events containing telomeric regions could be observed under conditions where the canonical DNA end joining pathway is compromised by mutations either in components of DNA-PK (DNA-PKcs, KU70, KU80), as well as in DNA Ligase IV or XRCC4, G. Iliakis' team investigated in detail the possibility that backup pathways of NHEJ exist that may gain prominence in NHEJ mutants and may cause the genomic and telomeric instability observed.
These results are likely to have relevance in radiation protection as they identify pathways of repair that may contribute to tumorigenesis. Understanding therefore of the function of these pathways as well as of their relative contribution to the repair of DNA DSBs is likely to contribute to our understanding of cancer development under various conditions and treatments.
To further define the role of DNA repair proteins in telomere function and telomere-driven radiosensitivity, Maria Blasco and her team (partner CNIO) has studied cells from mice deficient for the DNA repair proteins Ku86, DNA-PKcs, and PARP-1. Previous work of partner CNIO showed that both components of the DNA-PK complex, Ku86 and DNA-PKcs, had a role in telomere protection/capping in mouse cells. Recent results from CNIO extend such a role for Ku86 in telomere length maintenance and telomere capping tp human cells. A prediction from this finding was that DNA-PKcs deficiency should impact on the rate of telomere shortening in the context of the adult organism and during aging. Meanwhile, CNIO could demonstrate this for DNA-PKcs deficient mice, which show a faster rate of telomere loss with aging, which is coincidental with premature aging phenotypes. To further define the genetic interaction between telomerase and DNA repair proteins, CNIO generated mice doubly deficient for telomerase and either Ku86 or DNA-PKcs.
Telomerase-deficient Ku86 and DNA-PKcs null mice manifested accelerated loss of organismal viability compared to single telomerase-deficient mice correlating with proliferative defects and age-related pathologies but not with increased incidence of cancer. These results indicate that absence of telomerase and short telomeres in combination with DNA repair deficiencies accelerate the aging process without impacting on tumorigenesis.
The role of PARP in telomere protection and telomere length maintenance has been a matter of ongoing debate. CNIO has clarified this issue by generating mice doubly deficient in PARP-1 and telomerase. These animals were shown to have a normal rate of telomere shortening and loss of telomere capping in the context of telomerase deficiency and, accordingly, no effect of PARP-1 ablation on organismal viability could be detected. In addition, CNIO has collaborated with partner ENSL in studies on the putative role of PARP-2 at the mammalian telomere revealing a functional interaction between PARP-2 and the telomere binding protein TRF2.
TRF2 has been shown to interact with proteins involved in various DNA repair pathways, including non-homologous end joining (NHEJ). The current view that TRF2 acts as a repressor of DNA repair specifically at the telomeres is based on over-expression experiments in cultured cells. However, as yet, no viable mouse models with altered TRF2 function are available. To address this issue, CNIO has generated mice with increased expression of TRF2 under the keratin-5 promoter (K5-TRF2 mice). These mice exhibit a pronounced skin hyper-pigmentation, premature skin deterioration, as well as predisposition to develop neoplastic lesions in the skin. At the molecular level, keratinocytes from the skin of these mice show dramatic telomere shortening, loss of the telomeric G-strand overhang, as well as increased chromosomal instability. In addition, K5-TRF2 mice show an increased susceptibility to UV-irradiation and DNA cross-linking agents.
The obtained results indicate that the skin phenotype of K5-TRF2 mice, reminiscent of the human syndrome Xeroderma pigmentosum, results from a combination of defective DNA repair and short telomeres, as indicated by the further aggravation of this phenotype in the absence of telomerase. This work, thus defines TRF2 as a critical link between telomere function and DNA repair.
Fanconi anemia (FA) patients show accelerated telomere shortening. In order to get further insights in to the role of the FA pathway in telomere biology, Jordi Surralles and his team studied whether FANCD2, a key downstream factor in this pathway, binds to telomeres. They investigated whether, resembling NBS1, FANCD2 binds to telomeres during replication in a cell cycle regulated manner. To do so, they studied the co-localization of FANCD2 with the telomeric protein TRF1 and TRF2 in cells synchronized in different phases of the cell cycle. The results obtained in immortalized cells indicated that FANCD2 forms S-phase foci but that it does not bind to telomeres in S-phase. In 2004, it was reported that BLM interacts with FA proteins. Since it is known that BLM binds at telomeres in ALT cells, Jordi Surralles and his team studied whether FANCD2 colocalizes with TRF2 at PML bodies in ALT cells and also in primary cells.
The results indicates that FANCD2 binds to telomeres in late S/G2 phase in ALT cells but not in primary cells indicating that this binding is not due to the absence of telomerase and suggesting that FANCD2 plays a role in telomere maintenance by recombination. Thus, FANCD2 resembles other proteins involved in DNA repair and ionising radiation response such us ATM or NBS1 proving that all these proteins cross-talk at the molecular level to response to both radiation damage and telomere maintenance.
Jordi Surralles' objective was to study the architecture of the inter-phase nuclei from the telomere perspective and it response to ionising radiation. He found that telomeres have a defined 3D distribution pattern inside the interphase nucleus that is independent of histone H2AX. Telomeric distribution is maintained along cell cycle even after gamma-irradiation in a process independent of histone H2AX phosphorylation. Finally, some telomere aggregates corresponding to the telomeric region of short arm of acrocentric chromosomes are associated to nucleolus in a functional structure to allow the transcription of the ribosomic RNA. These aggregates suffer some changes along the cell cycle but are not altered by ionising radiation.
In the past, Maria Blasco and her team (partner CNIO) have shown that the telomerase reverse transcriptase component (Tert) may favour growth and survival independently of telomere length maintenance. They have generated two mouse models that over-express Tert under the keratin-5 promoter (K5-Tert) and the Lck promoter (Lck-Tert), thus targeting Tert expression to stratified epithelia or thymocytes, respectively. More recently, Maria Blasco and her team could show that mice constitutively overexpressing Tert in thymocytes and peripheral T cells (Lck-Tert mice) had a higher incidence of spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls. In addition, T-cell lymphomas in Lck-Tert mice were found to be more disseminated than those in wild-type controls and affected both lymphoid and nonlymphoid tissues. This dramatic effect of Tert over-expression was confirmed to be independent of the role of telomerase in telomere length maintenance. Finally, Tert constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes, although it resulted in greater chromosomal instability upon gamma-irradiation in Lck-Tert primary lymphocytes than in controls, suggesting that Tert over-expression may interfere with the cellular response to radiation-induced DNA damage.
In parallel, Maria Blasco and her team have studied K5-Tert mice, which over-express Tert in the skin and a variety of other tissues. In previous work, they reported that these mice showed increased tumorigenesis and a faster wound healing than wild-type controls. A more detailed analysis of this mouse model confirmed a higher incidence of both induced and spontaneous tumours, resulting in increased mortality during the first year of life but revealed that, in spite of this elevated tumour incidence and the initial lower survival, K5-mTert mice show an extension of the maximum lifespan of up to 3 months. This longer lifespan was found to be coincidental with a lower incidence of certain age-related degenerative diseases, mainly those related to kidney function and germline integrity. Importantly, these effects of telomerase over-expression were not related to differences in telomere length in aged K5-Tert mice compared to wild-type mice.
Furthermore, they could show that the telomerase RNA component, Terc, is necessary to mediate these effects of Tert over-expression. In contrast to K5-Tert mice that express Terc, K5-Tert mice lacking Terc do not show increased tumorigenesis or increased wound healing compared to wild-type controls. Indeed, Terc-deficient K5-Tert mice show a reduction in tumour growth compared to Terc null controls, indicating an inhibitory effect of Tert over-expression in the absence of Terc. These results suggest that the tumour-promoting effects of Tert over-expression require the formation of Tert/Terc complexes rendering Terc a potential target for telomerase-based anticancer therapies.
In the past, Maria Blasco and her team (partner CNIO) have shown that mice with critically short telomeres are highly sensitive to ionising radiation, i.e. show increased chromosomal instability upon gamma-irradiation. More recent results from a close collaboration between partners UAB and CNIO shed light on the mechanism underlying telomere-driven radiosensitivity. It was found that short telomeres re-join to double strand DNA breaks (DSBs), which in turn impairs proper DSB repair and, consequently, increases radiation-induced chromosomal instability.
This finding may be important for understanding the increased radiation sensitivity associated with age in humans, as well as provide a rationale for inter-individual differences to cytotoxic effects of radiation-based cancer therapy. In addition, partner CNIO found that mice with short telomeres were also hypersensitive to the alkylating agent MNU and oxidative stress. Together, these findings demonstrate a generalized hypersensitivity of mice with short telomeres to genotoxic agents and indicate that telomerase inhibitors may be combined with radiotherapy or other genotoxic agents to sensitise cancer cells to die. Related to this, partner CNIO is participating in ongoing studies on porphyrin-based telomerase inhibitors. Finally, partner CNIO contributed to studies showing that functional telomeres are important in mammalian meiotic synapsis and recombination. Of note shortened telomeres of late-generation telomerase-deficient mice impair meiotic synapsis and decrease recombination, in particular, in females. In response to telomere shortening, male germ cells mostly undergo apoptosis, whereas female germ cells preferentially arrest in early meiosis, suggesting sexually dimorphic surveillance mechanisms for telomere dysfunction during meiosis in mice. These findings might be of relevance for our understanding of sex-specific differences in radiation sensitivity.
More recently, using mouse models deficient for telomerase and the Ku86 telomere-binding protein, the CNIO team has performed a complete transcriptome analysis to assess the global effects of telomere shortening or telomere dysfunction in male germ cells. The transcriptional profile of germ cells with severe telomere shortening was similar to that of Ku86-deficient cells, suggesting that short telomeres trigger a DNA damage response similar to that triggered by Ku86 deficiency. The combination of short telomeres and Ku86 deficiency had an accumulative effect on the same set of genes.
In keeping with the particular importance of stem cell proliferation and differentiation for the outcome of radiotherapy, partner CNIO has participated in studies evaluating the impact of telomerase shortening on the stem cell compartment. The obtained results indicate that telomerase is required for differentiation of adult bone marrow stem cells. Interestingly, telomere attrition and the ensuing chromosomal instability dramatically impaired the in vitro proliferation of adult stem cells but did not affect the in vitro proliferative potential of embryonic stem cells. More recently, the CNIO team has determined the role of both telomerase and telomere length on epidermal stem cell behavior. In particular, mice with critically short and dysfunctional telomeres showed a defective mobilization of epidermal stem cells, which anticipated the fact that these mice are resistant to skin tumorigenesis protocols and show a premature aging of the skin. In turn, mice that over-expressed telomerase in the skin showed an augmented stem cell mobilization, also anticipating the fact that these mice are more prone to tumorigenesis.
In a further project, partner CNIO investigated whether the accelerated telomere shortening reported in Fanconi anemia (FA) hematopoietic cells relates to a direct role of the FA pathway in telomere maintenance. CNIO found that both hematopoietic (stem and differentiated bone marrow cells, B and T lymphocytes) and non-hematopoietic (germ cells, fibroblasts) Fancg null cells display normal telomere length, normal telomerase activity and normal chromosome end-capping, even in the presence of extensive cytogenetic instability induced by radiation. In addition, early-passage primary fibroblasts from FA patients of complementation group G as well as primary human cells with reduced FANCG expression (FANCG-shRNA IMR-90 knock-down cells) showed no signs of telomere dysfunction. These data, which show that accelerated telomere shortening in FA patients is not due to a role of FANCG at telomeres but rather a consequence of the disease, suggest that telomerase-based therapies could be useful prophylactic agents in FA aplastic anemia, by preserving their telomere reserve in the context of the disease.
During the progression of Telosens work, novel mechanisms of telomere maintenance were identified. Previous work of Maria Blasco and her team (partner CNIO) provide evidence for the implication of epigenetic mechanisms in the regulation of mammalian telomere length and function. Now, they have been successful in defining novel roles of SUV4-20 methyl transferases and the Retinoblastoma family of proteins (Rb, p107 and p130) in the interplay between heterochromatic features at telomeres and telomere length regulation.
In particular, loss of these proteins was found to increase the accessibility of heterochromatin facilitating the access of telomerase or other telomere-elongating activities to the chromosome end. Maria Blasco and her team further demonstrated a direct interaction between the Rb proteins and the H4K20 tri-methylating enzymes Suv4-20h1 and Suv4-20h2, suggesting a role of the Rb family in controlling H4-K20 tri-methylation by these novel histone methyltransferases. The observation that the Rb family is involved in maintaining overall chromatin structure and in particular that of constitutive heterochromatin including telomeric regions links telomere length, tumour suppression and the epigenetic definition of chromatin. More recently, a role for DNA methyltransferases has been shown by Blasco and colleagues in telomere length regulation and telomere recombination.
In particular, cells deficient in DNA methyltransferases showed a marked decreased in methylation of subtelomeric regions, which was coincidental with massive telomere elongation as well as with an increased telomeric recombination. The increased telomere recombination was also coincidental with increased presence of ALT-associated PML bodies (APB) in cells, which are a landmark for ALT mechanisms. These data suggest that DNA methylation acts repressing telomere recombination, therefore maintaining telomere integrity and telomere length.
Recent evidence has associated telomere instability to oncogenesis but the mechanisms and the significance for human cancers remains elusive. To explore the ability of telomere dysfunction to promote neoplastic transformation in defined human cellular systems, Eric Gilson and his team have developed human cellular systems to study the role of TRF2 in tumorigenesis and have shown that TRF2 dysfunction can trigger some of the steps involved in tumorigenesis. For the first time has been established a causal relationship between telomere instability and transformation of human cells. They have also shown that the effect of TRF2 in oncogenesis can occur in the presence of telomerase, suggesting that it could operate at late stages of human cancer progression.
The main result relevant to radioprotection of this task is the observation that, for a given cellular system i.e. immortalized fibroblasts, irradiation does not appear to contribute to transformation while telomere dysfunction does. This comparison is valid since approximately the same number of chromosome breaks and rearrangements were generated in either procedure. Therefore, telomere dysfunction appears to be more prone to trigger oncogenic events than gamma-irradiation, suggesting the existence telomere-specific events leading to transformation. Using extensive M-FISH analyses, Laure Sabatier and her team were unable to identify recurrent chromosome rearrangements, which could explain this difference. Therefore, they favour the idea that telomere dysfunction triggers an epigenetic reprogramming of the cell, which renders it more prone to acquire oncogenic properties. They are currently trying to characterize these epigenetic changes.
Regarding radioprotection, these results suggest that cells exhibiting damaged telomere are more prone to undergo transformation. One might predict that irradiation in circumstances that favour the targeting of telomeres (for examples with cells containing "fragile" telomeres, such as premalignant telomerase-negative cells), would favour transformation. Eric Gilson and his team are now testing whether this hold true for other cell types and for fully transformed cells.
In the past, Maria Blasco and her team have shown that mice with critically short telomeres are highly sensitive to ionising radiation, i.e. show increased chromosomal instability upon gamma irradiation. More recent results shed light on the mechanism underlying telomere-driven radiosensitivity. The CNIO team has determined the role of both telomerase and telomere length on epidermal stem cell behaviour (Flores et al., Science, 2005). In particular, mice with critically short and dysfunctional telomeres showed a defective mobilization of epidermal stem cells, which anticipated the fact that these mice are resistant to skin tumorigenesis protocols and show a premature aging of the skin. In turn, mice that over-expressed telomerase in the skin showed an augmented stem cell mobilization, also anticipating the fact that these mice are more prone to tumorigenesis.
It is known that DNA-PKcs is a key protein for the repair of DSBs through the NHEJ pathway and that DSBs can initiate gene amplification. The results obtained by Elena Giulotto and Chiara Mondello indicate that in human cells an impairment in DNA-PKcs function increases the proneness to DNA amplificaton. Promiscuous recombination events between broken ends, which are not immediately and correctly rejoined could trigger the amplification process. Alternatively, a defect in DNA-PKcs could cause an increase in gene amplification by altering the equilibrium between NHEJ and HR.
They also obtained human cell lines with good inhibition of DNA-ligase IV expression, however, the degree of protein level reduction was not related to sensitivity to IR, which was only slightly increased in some cell lines. This observation suggests that either low levels of the protein are sufficient for DSB rejoining or that other ligases, such as ligase III, can contribute to the repair of this kind of damage. On the other hand, the greatly reduced amplification ability of some ligase IV defective cell lines suggests that this protein may be essential for the recombination events accompanying gene amplification. To test this hypothesis they will analyze amplification in cell lines derived from mice in which the gene had been inactivated by homologous recombination.
The identification of genes playing a role both in radiation response and in gene amplification is relevant for the elucidation of the individual response to radiotheray. In fact, the rare cells surviving radiotherapy in tumours bearing mutations in genes which increase amplification frequency could be more prone to amplify oncogenes and drug resistance genes, giving rise to highly aggressive recurrences, in spite of their hyper-sensitivity to IR.
Telomeres are physical ends of chromosomes responsible for chromosomal end protection. There is a strong inter-dependence between telomere maintenance and cellular mechanisms that regulate DNA damage response. At least 17 mammalian genes involved in DNA damage response have, so far, been implicated in telomere maintenance and it is likely that the number of these genes will grow. The results generated by Predrag Slijepcevic and his team provide additional support for the link between telomere maintenance and DNA damage response mechanisms. The most significant finding, perhaps, is the potentially direct involvement of BRCA1 in telomere capping function. BRCA1 is a known tumour suppressor gene. Telomere maintenance also constitutes a powerful tumour suppressor mechanism. BRCA1 defective cells may, therefore, have failures in two tumour suppressor mechanisms.
The exact mechanisms through which BRCA1 exerts its tumour suppressor properties are not fully understood. It is now of interest to investigate the possibility that BRCA1 may exert, at least in part, its tumour suppression through effects on telomere maintenance. This finding is also relevant from the perspective of radiation protection. BRCA1 defects cause radiosensitivity and previously demonstrated link between radiosensitivity and telomere dysfunction provided by Dr Predrag Slijepcevic and his team has now been strengthened.
In addition, a range of primary cell lines from 11 patients that over-responded to radiotherapy were used by Dr. Predrag Slijepcevic and his team to demonstrate further the inter-dependence between telomere maintenance and DNA damage response mechanisms.
Collectively, above results provide support for the hypothesis that that telomere maintenance mechanisms and mechanisms of DNA damage response are tightly linked. A direct prediction from this hypothesis is that telomere maintenance may serve as a useful indicator of individual radiosensitivity and further research is required to test this link more stringently.
Mouse studies in the frame of this and previous EU project have determined that telomere length modulates radiosensitivity. Thus, telomerase KO mice with short telomeres (late generation) are more sensitive that telomere KO mice with long telomeres (earlier generation KO mice). In this study Jordi Surralles and his team studied whether cells from individuals with long telomeres are protected against radiation-induced chromosome damage. To avoid the effect of ageing on chromosome radiosensitivity, a total of 200 individuals of the same age were recruited and IR-induced chromosome damage was measured in those 10% individuals with shorter telomeres and 10% individuals with longer telomeres.
This partner found less radiation-induced chromosome damage in the subgroup of individuals with long telomeres. To their knowledge, this is the first study proving a relationship between human telomere length and radiation-induced chromosome fragility indicating that long telomeres protect from radiation induced DNA damage even. This effect was only observed when cells are irradiated at G1 phase of the cell cycle but not in G2 phase suggesting that the modulating effect of telomere length on radiosensitivity is cell cycle dependent. This study also provides a molecular link between diet and aging suggesting that high vegetable intake protects telomeres from an excessive shortening. This might be caused by higher levels of antioxidants such as folic acid in vegetables. This is supported with the phenotypic connexion between low intake of folic acid, short telomeres, and spina bifida. This study suggests the possibility that chemoprevention of telomere shortening might protect from radiation induced telomere damage. This hypothesis can be tested in mice animal models.
A simple and reliable procedure is now available, as a kit, to determine sperm DNA fragmentation. The integrity of sperm DNA is indeed being recognized as a new parameter of semen quality and a potential fertility predictor. However, it was not so far assessed as a routine part of semen analysis. A new improved technique to determine sperm DNA fragmentation has been established for human spermatozoa, being called the Sperm Chromatin Dispersion (SCD) test. This is a simple, fast, accurate and highly reproducible method for the analysis of sperm DNA fragmentation. It may be confidently estimated under the conventional bright-field microscope. Moreover, different degrees of DNA-nuclear damage can be detected as well as the discrimination of sperm nucleoids from other cell types.
Unlike other procedures, the SCD test can be used without the requirement of complex or expensive instrumentation. However, if desired, it could be visualized with automation. Finally, laboratory technicians can easily, quickly and reliably assess the test endpoints. Therefore, the improved SCD test could allow the routine screening of sperm DNA fragmentation in the basic andrology laboratory. This new technique is commercialised as a kit under the name Halosperm. Besides its application in the semen quality assessment and infertility studies, it has been demonstrated that ionising radiation exposure increases the frequency of spermatozoa with fragmented DNA, even for several months after the exposure. Biological dosimetry using this new parameter could be easily accomplished performing the SCD procedure.
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