SUMMARYSperm 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.

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.

SUMMARYSperm 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.

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.

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 H2 O2 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 H2 O2 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.