Costanza Calamai

In vitro manipulation of human spermatozoa during Assisted Reproductive Technology (ART) can induce several damages to sperm structure and functions. This study investigated the protective effects of Raphanus sativus seed extract and its active compounds on several sperm parameters during in vitro incubation and cryopreservation. Extracts from five seed-batches were characterized by HPLC-DAD-MS and ¹H-NMR, identifying sinapine and sinipic glycosides as the main characteristic compounds. Sperm DNA fragmentation (sDF) was detected by the Sperm Chromatin Dispersion test and LiveTUNEL. Excessive reactive oxygen species (ROS) production was detected by MitoSOX Red in viable spermatozoa. Caspase activity was detected by FLICA. Cryopreservation was conducted with two alternative freezing media. In vitro incubation with the extract protected against the loss of motility and reduced the induction of sDF, sperm ROS production, and caspase activity. Similarly, during cryopreservation, it allowed much better recoveries of sperm viability, motility, and DNA integrity by decreasing sperm ROS production with both freezing media. Sinapine and sinapic acid completely mimicked the protective effects of the whole extract during both in vitro incubation and cryopreservation, suggesting that they are included among the active principles. These findings support Raphanus sativus seed extract and its active compounds as candidates for inclusion in handling and freezing media for human spermatozoa in ART.

TUNEL, SCSA, SCD test and COMET assay are the current tests for detection of sperm DNA fragmentation (sDF) in clinical practice. These four tests are very different from each other for many aspects, possibly including the type of revealed damage. To verify how the same type of damage was revealed, we simultaneously detected sDF by the four tests before and after induction of sperm DNA breakage by cryopreservation and in vitro incubation. We found that all tests revealed the increase in sDF in both experimental conditions. However, when we pairwise compared the fold increases in induced sDF, we found poor (i.e., values below 0.5) Lin’s concordance correlation coefficients (CCCs) both during cryopreservation and in vitro incubation. The only exception was for SCD test/COMET assay where the CCCs were about 0.5 (cryopreservation: 0.456 (95% CI −0.071–0.784) and incubation: 0.523 (95% CI −0.018–0.827)). Bland–Altman plot analysis showed that TUNEL reveals the highest amounts of sDF during cryopreservation, whereas LiveTUNEL indicated that such damage is undergone by the viable sperm fraction. This is the first study comparing the four tests in detection of sperm DNA damage during cryopreservation and incubation.

Sperm oxidative stress has been extensively associated to male infertility. However, tests to detect this parameter have not been yet introduced in clinical practice and no definitive data are present on the extent of oxidative stress in male infertility. In this study, we used a novel and reliable flow cytometric method to reveal sperm ROS production in subfertile patients (n = 131) and in healthy donors (n = 31). Oxidative stress was higher in subfertile patients (14.22 [10.21–22.08]%) than in healthy donors (9.75 [8.00–14.90]% (p < 0.01)), but no correlation was found with age, semen quality or sDF. We also failed to detect an increase in sperm ROS production with semen viscosity or leukocytospermia, but a sharp impact of semen bacteria was evident (with bacteria: 31.61 [14.08–46.78]% vs. without bacteria: 14.20 [10.12–22.00]%, p < 0.01). Finally, after establishing a threshold as the 95th percentile in healthy donors, we found that 29% of subfertile patients exceeded this threshold. The percentage decreased to 25.56% when we excluded subjects with bacteriospermia and increased to 60.87% when only these patients were considered. In conclusion, 29% of subfertile patients showed an excessive sperm ROS production. Surprisingly, this parameter appears to be independent from routine semen analysis and even sDF determination, promising to provide additional information on male infertility.

Sperm DNA fragmentation (sDF) is a DNA damage able to predict natural conception. Thus, many laboratories added tests for the detection of sDF as an adjunct to routine semen analysis with specific indications. However, some points related to sDF are still open. The available tests are very different each from other, and a direct comparison, in terms of the prediction of reproductive outcomes, is mandatory. The proposed mechanisms responsible for sDF generation have not yielded treatments for men with high levels of sDF that have gained the general consent in clinical practice, thus requiring further research. Another relevant point is the biological meaning to attribute to sDF and, thus, what we can expect from tests detecting sDF for the diagnosis of male infertility. SDF can represent the “tip of iceberg” of a more extended and undetected sperm abnormality somehow impacting upon reproduction. Investigating the nature of such a sperm abnormality might provide novel insights into the link between sDF and reproduction. Finally, several studies reported an impact of native sDF on assisted reproduction technique outcomes. However, to fertilise the oocyte, selected spermatozoa are used where sDF, if present, associates with highly motile spermatozoa, which is the opposite situation to native semen, where most sDF associates with non-viable spermatozoa. Studies comparing the impact of sDF, as assessed in both native and selected spermatozoa, are needed.

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 10⁶/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure.

Cancer impairs spermatogenesis, whereas results on sperm DNA integrity are controversial and no data are available about sperm oxidative stress. In cancer patients, we detected sperm DNA fragmentation (sDF) and both viable (ROS production in viable sperm fraction/viable spermatozoa) and total (ROS production in viable sperm fraction/total spermatozoa) oxidative stress. We found that cancer (22.50 (17.00–26.75)%, n = 85) increased sDF with respect to the control groups in both normozoospermic subfertile patients (NSP) (12.75 (8.63–14.88)%, n = 52, p < 0.001) and in healthy donors (HD) (8.50 (7.00–14.00)%, n = 19, p < 0.001). The induction of viable oxidative stress (n = 96) with cancer was even higher: 36.60 (24.05–58.65)% versus 11.10 (8.63–14.90)% in NSP (p < 0.001) and 9.60 (8.00–14.03)% in HD (p < 0.001). Similar, albeit lower, differences were found for total oxidative stress. SDF sharply correlated to viable oxidative stress when we considered all subjects (cancer patients and controls) (r = 0.591, p < 0.001, n = 134), but no correlation was found when only cancer patients were studied (r = 0.200; p > 0.05, n = 63). In conclusion, cancer significantly increases sDF and sperm oxidative stress levels. Additional mechanisms to oxidative attack might be responsible for increased sDF in cancer patients. Because sperm oxidative stress might affect the outcomes of sperm cryopreservation, of cancer treatments and of sperm epigenoma, the detection of oxidative stress could be of help in managing the reproductive issues of cancer patients.