Maria San Gabriel

Antioxidants and sperm DNA damage: a clinical perspective

PurposeInfertile men possess substantially more sperm DNA damage than do fertile men, damage that may impact negatively on reproductive outcomes. In this era of assisted reproductive technologies there is mounting concern regarding the safety of utilizing DNA-damaged spermatozoa in this setting. Therefore, it is important to identify strategies that may reduce sperm DNA damage. The purpose of this review is to discuss the rationale for antioxidant therapy in men with sperm DNA damage and to evaluate the data on the efficacy of dietary and in vitro antioxidant preparations on sperm DNA damage.MethodsWe reviewed the literature on antioxidants and sperm DNA damage.ResultsTo date, the data suggest that dietary antioxidants may be beneficial in reducing sperm DNA damage, particularly, in men with high levels of DNA fragmentation. However, the mechanism of action of dietary antioxidants has not been established and most of the clinical studies are small. A beneficial effect of in vitro antioxidant supplements in protecting sperm DNA from exogenous oxidants has been demonstrated, however, the effect of these antioxidants in protecting sperm from endogenous ROS, gentle sperm processing and cryopreservation has not been established.

Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial

There is evidence from retrospective studies that varicocelectomy can improve sperm DNA damage in infertile men with a clinical varicocele. The objective of this prospective study was to examine further the effect of varicocelectomy on sperm chromatin and DNA integrity. We evaluated a consecutive series of infertile men (n = 25) who underwent microsurgical varicocelectomy for treatment of clinical varicocele. We examined conventional sperm parameters and sperm chromatin structure assay parameters (percentage DFI--DNA fragmentation index and percentage HDS--high DNA stainability, an index of chromatin compaction) before and 4 and 6 months after microsurgical varicocelectomy. Sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 18 ± 11% before surgery to 10 ± 5%, and 7 ± 3%, at 4 and 6 months after surgery respectively). Sperm chromatin compaction also improved significantly after surgery (percentage HDS decreased from 11 ± 7% before surgery to 8 ± 6%, and 7 ± 5%, at 4 and 6 months after surgery, respectively). Sperm concentration and progressive motility improved after surgery, although the differences were not statistically significant when compared with that before surgery. The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction. These findings support the concept that correction of a varicocele can improve spermatogenesis, particularly spermiogenesis (the stage in spermatogenesis where compaction and stability of the sperm DNA and chromatin occur).

Direct DNA Analysis with Paper-Based Ion Concentration Polarization

DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

Sperm head morphology is related to high deoxyribonucleic acid stainability assessed by sperm chromatin structure assay

OBJECTIVE To examine the relationship between sperm strict morphology and sperm chromatin integrity. DESIGN Prospective study. SETTING Infertility clinic. PATIENT(S) Eighty-seven consecutive semen samples from non-azoospermic men presenting for infertility evaluation and 6 samples from fertile donors. INTERVENTION(S) Assessment of standard semen parameters and sperm chromatin structure assay (SCSA) parameters (%DFI [DNA fragmentation index] and %HDS [high DNA stainability]). Evaluation of %HDS and %DFI after treatment with dithiothreitol (a thiol-reducing agent used to decondense sperm nuclei) was also undertaken. MAIN OUTCOME MEASURE(S) Relationship between sperm strict morphology defects and SCSA parameters (%DFI and %HDS). RESULT(S) We observed significant relationships between the percentage of normal sperm forms and both %HDS (r = -0.40) and sperm motility (r = 0.32). We also found significant relationships between sperm head defects and both %HDS (r = 0.40) and sperm concentration (r = -0.39). Sperm tail, midpiece, and neck defects were not significantly related to the SCSA parameters. Treatment of spermatozoa with dithiothreitol (to induce decondensation) resulted in a substantial increase in %HDS but no measurable change in %DFI. CONCLUSION(S) The observed relationship between sperm head defects and %HDS suggests that sperm head abnormalities may, in part, be due to incomplete sperm chromatin condensation.

Low amounts and high thiol oxidation of peroxiredoxins in spermatozoa from infertile men.

Seminal oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) and/or a decrease of antioxidant activity, promoting impaired sperm function. Peroxiredoxins (PRDX) are abundant in human semen and are important antioxidant enzymes, which act as ROS scavengers and modulators in ROS-dependent signaling. Our aim was to determine whether the levels of PRDX1 and PRDX6 and their oxidation on thiol groups are associated with a decrease in sperm motility and DNA integrity. We evaluated the sperm and seminal PRDX level in men (13 healthy controls, 15 men with clinical varicocele, and 17 men with idiopathic infertility). We assessed conventional semen parameters, sperm DNA integrity (by the sperm chromatin structure assay), lipid peroxidation in seminal plasma and spermatozoa (by the thiobarbituric acid reactive substances assay), and the amount and thiol oxidation of PRDX1 and PRDX6 (by immunoblotting). PRDXs were affected in seminal plasma (lower amounts) and in sperm samples (lower amounts and higher levels of thiol oxidation) characterized by lower sperm motility, higher lipid peroxidation, and sperm DNA damage. The thioloxidation ratio of PRDXs (thiol-oxidized PRDX/total PRDX) correlated negatively with sperm motility (total and progressive) and positively with sperm DNA damage and sperm lipid peroxidation. In conclusion, because of the lower amount of total PRDX1 and PRDX6 and the high thiol oxidation of these PRDXs, very little (less than 20%) protection due to PRDXs remains, and this is associated with impaired sperm function and poor DNA integrity and suggests an important role of PRDXs in the protection of human spermatozoa against oxidative stress.

Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage.

OBJECTIVES To examine the effects of lycopene on human sperm motility and DNA damage. DESIGN Prospective study. SETTING Andrology research laboratory. PATIENT(S) Twelve fertile donors. INTERVENTION(S) Preincubation of washed sperm suspensions with or without lycopene (2 or 5 micromol/L) followed by a 2-hour incubation with or without hydrogen peroxide (H2O2, 50 micromol/L). Assessments of sperm motility (percentage) and DNA fragmentation index (percent DNA fragmentation index) before and after incubation. MAIN OUTCOME MEASURE(S) Sperm motility (percentage) and sperm percent DNA fragmentation index. RESULT(S) Incubation of spermatozoa with H2O2 resulted in a significant decline in mean (+/-SD) percent sperm motility (28%+/-13% vs. 73%+/-4%, respectively) and a significant increase in percent DNA fragmentation index compared with samples incubated without H2O2 (29.8%+/-39.4% vs. 11.1%+/-14.6%, respectively). Pretreatment of samples with 5 micromol/L lycopene resulted in a significantly lower percent DNA fragmentation index than samples incubated without lycopene (8.0%+/-7.9% vs. 29.8%+/-39.4%, respectively). However, lycopene did not protect spermatozoa from the decline in motility after H2O2 treatment. CONCLUSION(S) The data suggest that preincubation of spermatozoa with lycopene offers protection against oxidative DNA damage in vitro. These data also highlight the differential protective effects of lycopene on sperm motility and sperm DNA integrity.

Sperm nuclear histone H2B: correlation with sperm DNA denaturation and DNA stainability

AIM To examine the relationship between sperm DNA damage and sperm nuclear histone (H2B) staining. METHODS We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone (H2B) staining and sperm chromatin integrity (assessed by sperm chromatin structure assay and expressed using the percentage of (i) DNA fragmentation index [% DFI] and (ii) high DNA stainability [% HDS)]) were evaluated. RESULTS Histone H2B immunocytochemistry demonstrated two nuclear staining patterns: (i) focal punctate staining; and (ii) diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men (7.7% +/- 4.6% vs. 1.6% +/- 1.2%, respectively, P < 0.01). We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm % DFI (r = 0.63, P < 0.01) and sperm %HDS (r = 0.63, P < 0.01). CONCLUSION The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.

Catalase can protect spermatozoa of FSH receptor knock-out mice against oxidant-induced DNA damage in vitro.

The aetiology of sperm DNA damage is likely multi-factorial with abnormal compaction of nuclear DNA, abortive apoptosis and oxidative stress implicated as potential causes of DNA damage. The objective of this study was to evaluate DNA damage in spermatozoa from wild-type (WT) and FSH receptor knock-out (FORKO) mice, compare the relative susceptibility of spermatozoa from these animals to oxidative DNA damage, and examine the protective effect of the antioxidant catalase on sperm DNA damage. Epididymal spermatozoa from FORKO mice (n = 5) and WT controls (n = 5) were extracted and incubated with or without catalase. Sperm DNA damage was assessed immediately after epididymal extraction (time 0 control) and following 2-h incubation at 37 °C. DNA damage was measured by the sperm chromatin structure assay and the results expressed as the %DNA fragmentation index or %DFI. Freshly retrieved epididymal spermatozoa from WT mice had a significantly lower mean (±SD) %DFI than that of FORKO mice (2.7 ± 1.8 vs. 6.4 ± 2.9%, p < 0.05). Prolonged (2-h) incubation of FORKO mice spermatozoa resulted in a significant increase in %DFI compared with the time 0 control (17.9 ± 9.2% vs. 6.4 ± 2.9%, respectively, p < 0.05) and the addition of catalase protected these spermatozoa from DNA damage (9.8 ± 4.1 vs. 17.9 ± 9.2%, respectively, p < 0.05). However, incubation of WT mice spermatozoa did not increase %DFI significantly (5.8 ± 5.0 vs. 2.7 ± 1.8, respectively, p > 0.05) and the addition of catalase (vs. no catalase) did not result in a significant reduction in %DFI (5.8 ± 5.0 vs. 7.7 ± 6.5%, respectively, p > 0.05). These data indicate that catalase may protect sperm nuclear DNA from oxidative stress in vitro. The data also demonstrate the differential susceptibility of WT and FORKO mice spermatozoa to oxidative stress.

Anti-sperm antibodies are not associated with sperm DNA damage: a prospective study of infertile men

Infertile men have poorer sperm DNA integrity than do fertile men, and this damage may contribute to reducing male reproductive potential. However, the etiology of this damage has not been fully characterized. We sought to examine the relationship, if any, between anti-sperm antibodies and sperm DNA damage in a consecutive series of non-azoospermic, infertile men in order to determine whether anti-sperm antibodies (ASAs) may contribute to sperm DNA damage. We conducted a prospective study on consecutive semen samples obtained from men (n=75) presenting for infertility evaluation. Sperm concentration, motility, strict morphology, ASA levels (by direct mixed agglutination reaction, expressed as the percentage of spermatozoa with IgG or IgA antibodies) and sperm DNA damage (by sperm chromatin structure assay) were evaluated. Mean (+/-SD) sperm concentration and progressive motility were significantly lower in ASA-positive (>40% of sperm coated with ASAs) compared to ASA-negative samples (23.4+/-13.1 x 10(6)/mL and 27+/-15% vs. 74.6+/-61.2 x 10(6)/mL and 46+/-18%, respectively, P<0.05). Sperm progressive motility was inversely correlated with the percentage of IgG-bound (r=-0.33) and IgA-bound spermatozoa (r=-0.25). In contrast, sperm %DNA fragmentation index and percent normal forms were not significantly different in ASA-positive compared to ASA-negative samples (17.5+/-17.9% and 7.5+/-3.0% vs. 17.4+/-13.5% and 6.5+/-2.6%, respectively). The data indicate that ASAs are not associated with sperm DNA damage and suggest that ASAs are unlikely to have a significant direct or indirect effect on sperm DNA integrity.

Paper-based sperm DNA integrity analysis

We demonstrate a rapid, sensitive, and low cost paper-based approach for sperm chromatin integrity analysis that quantifies both DNA fragmentation and packaging. The paper-based test provides identical clinical outcome as the gold standard sperm chromatin structure assay. This technology is fast, simple, and suitable for broad application in clinics.