Análisis de la fragmentación del ADN de espermatozoides humanos mediante el test de dispersión de la cromatina espermática : origen, causas e impacto en reproducción asistida

[Resumen] Los hombres infértiles presentan una mayor proporción de espermatozoides con ADN fragmentado que los hombres fértiles, aunque un cierto porcentaje de estas células siempre está presente en todas las muestras seminales. Esta tesis se centra en el estudio de ciertas cuestiones básicas y clínicas acerca de la fragmentación del ADN espermático. Desde el punto de vista de la investigación básica, nuestros resultados indican que el estrés oxidativo se relaciona con la fragmentación del ADN observada en los espermatozoides. Además, la matriz proteica nuclear también está afectada en aquellos espermatozoides con el ADN fragmentado. Con respecto al impacto clínico, constatamos que el cáncer y la infección genitourinaria por Chlamydia trachomatis y Mycoplasma sp. incrementan la frecuencia de espermatozoides con el ADN fragmentado, lo cual podría contribuir a la infertilidad observada en estos pacientes. En relación al efecto en reproducción asistida, hemos observado que, aunque la fragmentación del ADN espermático afecta negativamente a la probabilidad de obtener un embarazo, un ovocito de buena calidad podría superar el impacto negativo de la fragmentación. Por último, en el contexto de la toxicología reproductiva, el estudio dinámico de la fragmentación del ADN espermático fue capaz de revelar daño latente en el espermatozoide, que no era evidente inicialmente tras la exposición a niveles tóxicos de ciertos agentes.[Resumo] Os homes infértiles presentan unha maior proporción de espermatozoides co ADN fragmentado que os homes fértiles, aínda que certa porcentaxe destas células sempre está presente en todas as mostras seminais. Esta tese céntrase no estudo de certas cuestións básicas e clínicas acerca da fragmentación do ADN espermático. Dende o punto de vista da investigación básica, os nosos resultados indican que o estrés oxidativo se relaciona coa fragmentación do ADN observada nos espermatozoides. Ademais, a matriz proteica nuclear tamén está afectada naqueles espermatozoides co ADN fragmentado. Con respecto ao impacto clínico, constatamos que o cancro e a infección por Chlamydia trachomatis e Mycoplasma sp. producen un aumento na frecuencia de espermatozoides co ADN fragmentado, o cal podería contribuír á infertilidade observada nestes pacientes. En relación ao efecto en reprodución asistida, observamos que, aínda que a fragmentación do ADN espermático afecta negativamente á probabilidade de obter un embarazo, un ovocito de boa calidade podería superar o impacto negativo da fragmentación. Por último, no contexto da toxicoloxía reprodutiva, o estudo dinámico da fragmentación do ADN espermático foi capaz de revelar dano latente no espermatozoide que non era evidente inicialmente tras a exposición a niveles tóxicos de certos axentes.[Abstract] Infertile men have higher proportion of sperm with fragmented DNA than fertile men, although certain percentage of these cells is always present in all seminal samples. This thesis focuses on the study of certain basic and clinical questions about sperm DNA fragmentation. From the point of view of the basic research, our results show that oxidative stress is associated with the DNA fragmentation observed in the sperm. Moreover, the nuclear protein matrix is also affected in sperm with fragmented DNA. Regarding the clinical impact, we found that cancer and infection caused by Chlamydia trachomatis and Mycoplasma sp. increase the frequency of sperm with fragmented DNA, which could contribute to infertility observed in these patients. In relation to the effect on assisted reproduction, we observed that, although sperm DNA fragmentation negatively affects the probability of a pregnancy, oocyte quality could overcome the negative impact of fragmentation. Finally, within the context of reproductive toxicology, the dynamic assessment of sperm DNA fragmentation may reveal cryptic damage in the sperm, which was not evident immediately after the exposure to toxic levels of certain agents

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the beta-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after beta-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different beta-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis