Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

The impact of five years storage/biobanking at -80°C on mouse spermatozoa fertility, physiology, and function.

BACKGROUND: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs. OBJECTIVES: The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. MATERIALS AND METHODS: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. RESULTS: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2 . DISCUSSION: Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. CONCLUSION: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

Shape based kinetic outlier detection in real-time PCR.

BACKGROUND: Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. RESULTS: Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. CONCLUSION: Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

Cancer mortality among men occupationally exposed to dichlorodiphenyltrichloroethane.

Several studies have evaluated cancer risk associated with occupational and environmental exposure to dichlorodiphenyltrichloroethane (DDT). Results are mixed. To further inquire into human carcinogenicity of DDT, we conducted a mortality follow-up study of 4,552 male workers, exposed to DDT during antimalarial operations in Sardinia, Italy, conducted in 1946 to 1950. Detailed information on DDT use during the operations provided the opportunity to develop individual estimates of average and cumulative exposure. Mortality of the cohort was first compared with that of the Sardinian population. Overall mortality in the cohort was about as expected, but there was a deficit for death from cardiovascular disease and a slight excess for nonmalignant respiratory diseases and lymphatic cancer among the unexposed subcohort. For internal comparisons, we used Poisson regression analysis to calculate relative risks of selected malignant and nonmalignant diseases with the unexposed subcohort as the reference. Cancer mortality was decreased among DDT-exposed workers, mainly due to a reduction in lung cancer deaths. Birth outside from the study area was a strong predictor of mortality from leukemia. Mortality from stomach cancer increased up to 2-fold in the highest quartile of cumulative exposure (relative risk, 2.0; 95% confidence interval, 0.9-4.4), but no exposure-response trend was observed. Risks of liver cancer, pancreatic cancer, and leukemia were not elevated among DDT-exposed workers. No effect of latency on risk estimates was observed over the 45 years of follow-up and within selected time windows. Adjusting risks by possible exposure to chlordane in the second part of the antimalarial operations did not change the results. In conclusion, we found little evidence for a link between occupational exposure to DDT and mortality from any of the cancers previously suggested to be associated.