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The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel-Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel-Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine., (© 2020 Wiley-VCH GmbH.)

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2 hr, T6 hr and T24 hr of sperm thawing) were used to establish cut-off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2 hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF-T 0 by the increase in the damage registered during the first 2 hr of incubation (r-SDF-T 0-2 ), an index of RCD = 22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut-off values could be used to characterise donors with high chromatin resistance to damage when meeting the above-established criteria., (© 2018 Blackwell Verlag GmbH.)

The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility., (© 2014 Blackwell Verlag GmbH.)

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7-dibrom-4-hydroxy-mercury-fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two-dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double- and single-strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele., (© 2013 Blackwell Verlag GmbH.)

The rate of increase of sperm DNA fragmentation (rsDF) in fresh and frozen-thawed and processed sperm samples after a density gradient for sperm selection was analysed after 0, 0.5, 1.5, 4.5, 6, 24, 48 and 72 h of incubation at 37 °C, in five donors with proven fertility. The results showed that: (i) sperm DNA fragmentation (sDF) at baseline in fresh samples (14.3 ± 3.3) was lower than that obtained after freeze-thawing and selection (19.4 ± 4.1), significant differences; (ii) After 6 h of incubation the mean sDF in fresh samples (24.2 ± 10.2) was significantly lower than that in frozen-thawed samples (45.3 ± 7.1); (iii) Subsequently, the rsDF in fresh semen samples was 1.6% per h after 6 h of incubation, while after thawing and selection the rsDF was 4.3% per h; The tendency to increase in sDF showed high R(2) values (R(2) = 0.90) for exponential functions in case of fresh samples, whereas R(2) values for linear functions were higher after sperm selection (R(2) = 0.97). These results indicate that differences in sperm DNA fragmentation dynamics before and after storage are an important issue that must be considered for storage of sperm to be used for artificial reproduction techniques., (© 2011 Blackwell Verlag GmbH.)