María José Amengual

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.

Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm

OBJECTIVE To investigate the relationship between the protamine 1 to protamine 2 (P1/P2) ratio and the rate of sperm DNA fragmentation in sperm samples from human males with proven fertility and three different cohorts of male patients. DESIGN P1/P2 ratio was analyzed using acid-urea polyacrylamide acid-urea gels electrophoresis (PAGE). Sperm DNA fragmentation using sperm chromatin dispersion methodology was analyzed after 0, 4, 8, and 24 hours of incubation at 37°C. SETTING University medical school and hospital. PATIENT(S) A total of 32 human males: six with proven fertility, seven carriers of chromosome reorganizations, nine clinical varicocele patients, and ten subclinical varicocele patients. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) P1/P2 ratio, sperm DNA fragmentation (SDF) and the rate of sperm DNA fragmentation (rSDF). RESULT(S) P1/P2 ratio correlated with SDF and rSDF. Statistical differences were detected between fertile controls and patients for the three pathologies studied. rSDF yielded information that differed from baseline SDF. No differences were detected for P1/P2 ratio among patient groups, in reference to the three pathologies studied. CONCLUSION(S) SDF and rSDF correlates with P1/P2 ratio in human sperm, and statistical differences were detected when fertile controls were compared with three different cohorts of patients.

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.

Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor

It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL.

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.

Differential Clustering of Sperm Subpopulations in Infertile Males With Clinical Varicocele and Carriers of Rearranged Genomes

Some methods for determining sperm DNA fragmentation, such as the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion test (SCD), provide additional information about particular subgroups of spermatozoa with specific irregularities. Thus, SCSA recognizes a specific sperm subpopulation, the high-DNA stainability sperm subpopulation (HDS), and SCD recognizes the so-called DNA-degraded sperm (DDS) subpopulation. Although some studies associate the presence of these subpopulations with specific aspects related to infertility, the relationship between both sperm subpopulations and their preponderance in specific clinical groups of infertile males has not been extensively investigated. In this study, HDS and DDS subpopulations were determined in a total of 37 human males: 8 males with proven fertility, 9 infertile males with asthenoteratozoospermia, 10 carriers of chromosomal reorganizations, and 10 infertile males with clinical varicocele. Results showed a significant increase of the DDS subpopulation (P < .001) in both the varicocele patient (16.85 ± 7.24) and carrier of rearranged genome (11.6 ± 5.23) groups, but not in patients with asthenoteratozoospermia (3.88 ± 1.55) or fertile donors (2.62 ± 1.68). No statistical differences were detected for the HDS subpopulation (P = .542), but the highest values were found in the varicocele and rearranged-genome groups. However, no correlation between the HDS and DDS subpopulations were found (r = 0.196; P = .244), suggesting that both represent a different class of sperm subpopulation in the ejaculate. A significant increase in HDS, and especially DDS, can be associated with the presence of varicocele or the rearrangement of chromosomes. Specific diagnostic tests to confirm the diagnosis must be performed in patients with increased DDS and HDS values.

Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

Abstract Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.

Characterization of Nuclease Activity in Human Seminal Plasma and its Relationship to Semen Parameters, Sperm DNA Fragmentation and Male Infertility.

PURPOSE Some studies have shown that complementary biomarkers are needed in semen analysis to provide a more accurate diagnosis for couples with infertility problems. To our knowledge no study has been done to determine the relationships among nuclease activity in seminal plasma, semen parameters, sperm DNA fragmentation and male infertility. MATERIALS AND METHODS A total of 94 semen samples were collected according to WHO 2010 semen analysis parameters. Samples were analyzed using the single radial enzyme diffusion method for nuclease activity in seminal plasma, and alkaline and neutral Comet assay for sperm DNA fragmentation. Samples were obtained from 11 fertile donors with proven fertility, 17 patients with normozoospermia in an infertile couple, and 16 patients with asthenozoospermia, 19 with teratozoospermia, 21 with asthenoteratozoospermia and 10 with azoospermia. RESULTS Nuclease activity analyzed in seminal plasma was higher in patients than in controls. It correlated with sperm motility and morphology, and sperm DNA fragmentation measured by the alkaline Comet assay. No correlation with sperm DNA fragmentation was measured by the neutral Comet assay. ROC curves to determine male infertility revealed 0.658 sensitivity, 0.727 specificity and 0.705 cm(2) AUC for the single radial enzyme diffusion method, 0.918, 1 and 0.994 cm(2) for the alkaline Comet assay, and 0.917, 0.250 and 0.373 cm(2), respectively, for the neutral Comet assay. CONCLUSIONS Nuclease activity in seminal plasma corrected by sperm count is a good variable to predict male infertility. Results indicate that it could be a useful complementary parameter for male infertility diagnosis.

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 H2O2 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 H2O2 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.

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+.