Sperm telomere length in motile sperm selection techniques: A qFISH approach.

Several studies have associated telomere shortening with alterations in reproductive function. The objective of the present study was to determine telomere length (TL) in spermatozoa selected by either density-gradient centrifugation (DGC) or swim-up. The analysis of TL was performed using quantitative fluorescent in situ hybridisation (qFISH) using PNA probes in combination with a chromatin decompaction protocol in sperm cells. Results of TL were 24.64 ± 5.00 Kb and 24.95 ± 4.60 Kb before and after DGC, respectively, and 19.59 ± 8.02 Kb and 20.22 ± 5.18 Kb before and after swim-up respectively. Sperm selected by DGC or swim-up did not show any significant differences in TL as compared to nonselected sperm (p > .05). Negative correlations between TL and sperm motility (r = -.308; p = .049) and concentration (r = -.353; p = .028) were found. Furthermore, exposure of sperm to increasing concentrations of hydrogen peroxide during incubation resulted in a reduction in TL. These data indicate that oxidative stress may be one of the main factors involved in the reduction of TL in sperm. Preliminary clinical results from patients included in this study indicate that TL was shorter in spermatozoa from couples who never achieved a pregnancy compared to couples who did achieve at least one natural pregnancy (p < .05); however, the clinical utility of this biomarker still needs to be confirmed in further studies.

Spatial-temporal Variations of Bovine Tuberculosis Incidence in France between 1965 and 2000.

We analysed the spatiotemporal variations of bovine tuberculosis (bTB) incidence between 1965 and 2000 in France at the department level (95 areas). Using a Bayesian space-time model, we studied the association between the evolution of bTB incidence and changes of cattle population structure and of herd management practices. Several spatiotemporal hierarchical Bayesian models were compared, and the deviance information criterion was used to select the best of them. Southern France remained a high-risk area over the analysed period, whereas central and western regions were low-risk areas. Besides the frequency of tuberculin skin testing (fixed according to bTB incidence in the preceding years), four factors were associated with an increased risk of bTB: the average herd density and size, the percentage of dairy cows in the cattle population, and the percentage of permanent grassland in cultivated surfaces area. These four factors are linked to the progressive professionalization and specialization of cattle farming, with the disappearance of family farms and of the intensification of breeding systems (especially in dairy farms after the application of the milk quota system in the 1980s). Both trends probably played a significant role in reducing the risk of bTB in France between 1965 and 2000, besides mandatory detection and control procedures.

Nuclear degraded sperm subpopulation is affected by poor chromatin compaction and nuclease activity.

There is an interest in the nuclear degraded sperm subpopulation because, although it is present in a low percentage in all semen samples, patient groups such as varicocele and rearranged genome carriers show high levels of these degraded spermatozoa. This study is designed with two objectives in mind: first, incubations of H2 O2 and nuclease on DTT-treated and untreated samples to show the aetiology of this subpopulation and second, assessment of the correlation between the protamine ratio and nuclear degraded spermatozoa. A very high increase in the nuclear degraded subpopulation has been found with nuclease incubation, and it is even higher when it has been merged with nuclear decompaction using DTT. Alternatively, incubation with H2 O2 with and without DTT did not show such a significant increase in nuclear degraded spermatozoa. The protamine ratio correlated with this subpopulation, showing, in patients, that poor nuclear compaction would turn the sperm susceptible to degradation. Then, the assessment of nuclear degraded spermatozoa might not be only a measure of DNA degradation but also an indicator of chromatin compaction in the spermatozoa. Different patient groups would fit this model for sperm nuclear degradation, such as varicocele patients, who show a high percentage of immature spermatozoa and nuclear degraded spermatozoa, and reorganised genome carriers, where reorganisation might also cause poor chromatin compaction on the sperm nucleus.

Field-induced sublimation in perfect two-dimensional colloidal crystals.

Phase transitions in two-dimensional (2D) systems are of considerable fundamental and practical importance. However, the kinetics of these processes are difficult to predict and understand, even in simple systems for which equilibrium states are properly described, owing to the difficulty of studying crystallites with single-particle resolution and free of defects. Here we introduce an alternative method for the sublimation of 2D colloidal crystallites by a sudden induction of repulsive forces between the particles. The sublimation kinetics, studied in real space by microscopy and by computer simulations, shows a scaling behavior that suggests a universal mechanism fundamentally different from the one usually accepted for thermal sublimation. The universal behavior found for the early stages of the process may be useful for understanding the dynamic features of particle systems at liquid interfaces and for designing technological applications without the need of performing extensive experimental studies.

Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men.

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 µg of vitamin B9; 50 µg of selenium; 1 µg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.

First successful double-factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening.

Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-gene disorders over the last 20 years. The aim of this work was to perform a double-factor preimplantation genetic diagnosis (DF-PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF-PGD approach in which two blastomeres from each cleavage-stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non-affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short-comparative genomic hybridization (CGH). The improved DF-PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single-gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.

Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups.

BACKGROUND: The analysis of sperm DNA fragmentation has become a new marker to predict male infertility, and many techniques have been developed. The sperm Comet assay offers the possibility of differentiating single- and double-stranded DNA (ssDNA and dsDNA) breaks, which could have different effects on fertility. The objective of this study was to perform a descriptive characterization of different groups of patients, such as those with asthenoteratozoospermic (ATZ) with or without varicocele, oligoasthenoteratozoospermic (OATZ) or balanced chromosome rearrangements, as compared with fertile donors. The Comet assay was used to investigate sperm samples for ssDNA and dsDNA breaks. METHODS AND RESULTS: The analysis of alkaline and neutral Comet assays in different groups of patients showed different sperm DNA damage profiles. Most fertile donors presented low values for ssDNA and dsDNA fragmentation (low-equivalent Comet profile), which would be the best prognosis for achieving a pregnancy. OATZ, ATZ and ATZ with varicocele presented high percentages of ssDNA and dsDNA fragmentation (high-equivalent Comet assay profile), ATZ with varicocele being associated with the worst prognosis, due to higher levels of DNA fragmentation. Rearranged chromosome carriers display a very high variability and, interestingly, two different profiles were seen: a high-equivalent Comet assay profile, which could be compatible with a bad prognosis, and a non-equivalent Comet assay profile, which has also been found in three fertile donors. CONCLUSIONS: Comet assay profiles, applied to different clinical groups, may be useful for determining prognosis in cases of male infertility.

Dynamics of sperm DNA fragmentation in patients carrying structurally rearranged chromosomes.

This investigation was conducted to assess the baseline level of sperm DNA fragmentation (SDF) in a cohort of patients presenting chromosomal rearrangements (nine reciprocal translocations and two inversions). In a separate experiment, a dynamic analysis to calculate the rate of SDF (rSDF), after a varying period of sperm storage (0 h, 1 h, 4 h, 8 h and 24 h) at 37 °C, was performed. Results were compared with eight fertile donors. Different experimental approaches to assess SDF, such as terminal transferase dUTP nick-end labelling (TUNEL), sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCDt), were used. No differences for the baseline level of SDF were found. Carriers of reorganized genomes showed statistically higher levels of SDF than did control donors (p = 0.025 for TUNEL; p = 0.022 for SCSA; p = 0.014 for SCDt). However, 54.5% (6/11) of the patients presented values similar to those of control donors. There was no significant difference in rSDF (p = 0.34). Nevertheless, the results suggest that a high variability for SDF and rSDF exists in these patients. Routine analysis of SDF and rSDF should be considered in patients presenting rearranged genomes to determine fertility status for assisted reproductive techniques (ART) purposes.

Whole-chromosome aneuploidy analysis in human oocytes: focus on comparative genomic hybridization.

The study of aneuploidy in human oocytes, discarded from IVF cycles, has provided a better understanding of the incidence of aneuploidy of female origin and the responsible mechanisms. Comparative genomic hybridization (CGH) is an established technique that allows for the detection of aneuploidy in all chromosomes avoiding artifactual chromosome losses. In this review, results obtained using CGH in single cells (1PB and/or MII oocytes) are included. The results of oocyte aneuploidy rates obtained by CGH from discarded oocytes of IVF patients and of oocyte donors are summarized. Moreover, the mechanisms involved in the aneuploid events, e.g. whether alterations occurred due to first meiotic errors or germ-line mitotic errors are also discussed. Finally, the incidence of aneuploid oocyte production due to first meiotic errors and germ-line mitotic errors observed in oocytes coming from IVF patients and IVF oocyte donors was assessed.

Sperm DNA integrity and meiotic behavior assessment in an infertile male carrier of a 9qh+++ polymorphism.

Although several reports on male infertility suggest a relationship between chromosome 9 polymorphisms and infertility, the effects on the phenotype have not been extensively reported. In this study, an infertile patient was found to carry a 9qh+++ chromosome. The flow cytometric TUNEL assay and SCD test have been applied to characterize sperm DNA integrity. In order to assess its meiotic behaviour, synapsis, recombination, and aneuploidy, analyses have been also performed. Sperm DNA fragmentation (SDF) was 77.81% and 87% for the TUNEL and SCD tests, respectively. Ninety-two percent of pachytene cells analyzed showed meiotic abnormalities. The mean number of MLH1 foci per pachytene in the control group was higher (49) than the mean found in the 9qh+++ patient (38) (P < .0001). In spermatozoa, significant increases of disomy rates were observed for chromosome 18 and for the sex chromosomes (P < .0001). These disturbances could be present in other male carriers of a less marked 9qh+.

Analisis estadístico de 134 casos consecutivos de ganglio centinela en el melanoma maligno cutáneo en las islas baleares. Utilidad terapeútica de la biopsia selectiva ganglionar e importancia d ela localización anatómica del tumor primario (2000-2006) / Statistic analyisis of 134 consecutive cases of sentinel lymph node in cutaneous malign melanoma in baleares islands. Therapeutic utility of selective sentinel limph node biopsy biopsy and relevance of anatomic localization of primary tumour (2000-2006)

La biopsia selectiva del ganglio centinela en el melanoma maligno cutáneo nos permite una apropiada estadificación, evitando innecesarias linfadenectomías y verificar que las ideas clásicas de las vías de drenaje linfático no se corresponden con la realidad, existiendo una gran variabilidad según su localización anatómica, y que el Breslow es el factor pronóstico más importante para predecir la supervivencia junto a otros en estos pacientes. La cirugía es pues necesaria para una adecuada clasificación de la enfermedad y planificar su tratamiento, extirpar el tumor primitivo y posibles metástasis de las áreas ganglionares y eliminar metástasis viscerales disminuyendo la masa tumoral para facilitar otros procedimientos. El motivo de este trabajo es observar si existe correlación entre ellos e intentar obtener los resultados que tras una apropiada detección gammagráfica (con un 100% de aciertos en la localización) y quirúrgica del ganglio centinela, determinando si existen diferencias respecto al número de drenajes y de ganglios centinelas extraídos que justifiquen las variaciones encontradas (AU)

The selective biopsy of the sentinel lymph node in the cutaneous malignant melanoma allows us to make an appropriate staging, avoiding unnecessary lymphadenectomies and verify that the classical ideas of lymphatic drainage ways do not correspond with reality, as there is great variability according to anatomic localisation, and the fact that Breslow thickness is the most important prognostic factor for predicting survival along with others in these patients. Thus, surgery is necessary for an adequate classification of the disease and to plan its treatment, remove the primitive tumour and possible metastasis of the ganglionic areas and eliminate visceral metastasis diminishing the tumour mass to facilitate other procedures. The reason for this work is to find out whether there is a correlation between them and try to obtain the results after an appropriate gammagraphic etection (with 100% correct choice in the localisation) and surgery of the sentinel lymph node and determine if there are differences with respect to the number of drainages and extracted sentinel lymph nodes that justify the variations found (AU)

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application.

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Metástasis digestivas por melanoma maligno cutáneo. Análisis de dos casos / Metastases for cutaneus malignant melanoma. Analysis of two cases

Los sitios metastáticos mas frecuentes del melanoma maligno cutáneo son pulmones, hígado y encéfalo. Las metástasis intestinales por melanoma son raras siendo el intestino delgado el lugar mas afectado, por lo que su diagnóstico es difícil preoperatoriamente, junto con una clínica por lo general anodina, pudiendo presentarse como un cuadro anémico crónico, oclusivo o de perforación, por lo que suele diagnosticarse en el acto operatorio. Presentamos 2 casos de metástasis digestivas en pacientes intervenidos de melanoma maligno cutáneo que debutaron respectivamente con metástasis en duodeno y en ileon erminal. Este último reintervenido por metástasis mesentérica, muy cercana de la resección anterior. Ambos están en este momento con tratamiento oncológico y con estudios de extensión dentro de la normalidad (AU)

The most frequent metastasic sites of cutaneous malignant melanoma are lungs, liver and brain. Intestinal metastases by melanoma are rare. Most cases are located in small intestine, and its preoperative diagnosis is often difficult. Clinical symptoms are often anodyne, frequently appearing as a chronic, occlusive or anemic syndrome, or as a perforation, and to a late diagnosis after surgery. We report here 2 cases of digestive metastases of cutaneous malignant melanoma in duodenum and terminal ileum. The most frequent metastasic sites of cutaneous malignant melanoma are lungs, liver and brain. ntestinal metastases by melanoma are rare. Most cases are located in small intestine, and its preoperative diagnosis is often difficult. Clinical symptoms are often anodyne, frequently appearing as a chronic, occlusive or anemic síndrome, or as a perforation, and to a late diagnosis after surgery. We report here 2 cases of digestive metastases of cutaneous malignant melanoma in duodenum and terminal ileum. This last one underwent further surgery for mesentheric metastasis in the vicinity of previous surgery. Both patients are at the moment on oncology treatment and extension studies have shown so far no further metastasis (AU)

The organisation and functioning of Veterinary Services: results of a 2005 survey of Member Countries of the World Organisation for Animal Health.

A questionnaire was sent to the 167 Member Countries of the World Organisation for Animal Health (OIE) in 2004 and 2005. The organisation and functioning of national Veterinary Services were analysed based on the responses from 85 of these countries. Leaving aside variations between countries, Veterinary Services are very involved in animal health and food safety controls at farm level (including animal feed), and during primary and secondary processing, whether alone or in conjunction with other services. At the lower end of the chain, namely distribution and the food service industry, responsibilities tend to be more widely shared. Veterinary Services have a central responsibility in international trade in animals and animal products. The main weaknesses in the chain of controls concern the logistical and financial resources of Veterinary Services, and insufficient involvement of livestock producers and even of field veterinarians. The many recent reforms are tending to provide a more consistent, integrated approach to animal health and food safety controls 'from the stable to the table'.

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.

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.