Severe sperm DNA fragmentation may persist for up to 3 years after cytotoxic therapy in patients affected by Hodgkin lymphoma and non-Hodgkin lymphoma.

STUDY QUESTION: Does sperm DNA recover from damage in all men after 2 years from the end of cytotoxic treatments? SUMMARY ANSWER: The current indication of 2 years waiting time for seeking natural pregnancy after cytotoxic treatment may not be adequate for all men, since severe sperm DNA damage is present in a proportion of subjects even after this timeframe. WHAT IS KNOWN ALREADY: Data in the literature on sperm DNA fragmentation (SDF) in lymphoma patients after cytotoxic treatments are scarce. The largest longitudinal study evaluated paired pre- and post-therapy (up to 24 months) semen samples from 34 patients while one study performed a longer follow-up (36 months) in 10 patients. The median/mean SDF values >24 months after therapy did not show significant differences but the studies did not explore the proportion of patients with severe DNA damage and the analysis was done on frozen-thawed samples. STUDY DESIGN, SIZE, DURATION: In this study, 53 Hodgkin lymphoma (HL) and 25 non-Hodgkin lymphoma (NHL) post-pubertal patients were included over a recruitment period of 10 years (2012-2022). Among them, 18 subjects provided paired semen samples for SDF analysis at the three time points. SDF was evaluated in patients before (T0) and after 2 (T2) and 3 years (T3) from the end of, cytotoxic treatments (chemotherapy alone or in combination with radiotherapy). A cohort of 79 healthy, fertile, and normozoospermic men >18 years old served as controls (recruited between 2016 and 2019). PARTICIPANTS/MATERIALS, SETTING, METHODS: SDF was evaluated on fresh semen samples (i.e. spermatozoa potentially involved in natural conception) from patients and controls using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled with flow cytometry. SDF median values were compared between groups: (i) HL and NHL patients versus controls at the three time points; (ii) HL versus NHL patients at baseline; and (iii) patients at T0 versus T2 and T3. Severe DNA damage (SDD) was defined for SDF levels above the 95th percentile of controls (50%) and the proportion of patients with SDD at all time points was established. MAIN RESULTS AND THE ROLE OF CHANCE: At T0, patients displayed higher median SDF than controls, reaching statistical significance in the NHL group: 40.5% [IQR: 31.3-52.6%] versus 28% [IQR: 22-38%], P < 0.05. Comparing SDF pre-treatment to that post-treatment, HL patients exhibited similar median values at the three time points, whereas NHL showed significantly lower values at T3 compared to T0: 29.2% [IQR: 22-38%] versus 40.5% [IQR: 31.3-52.6%], P < 0.05. The proportion with SDD in the entire cohort at T2 was 11.6% and 13.3% among HL and NHL patients, respectively. At T3, only one in 16 NHL patients presented SDD. LIMITATIONS, REASONS FOR CAUTION: TUNEL assay requires at least 5 million spermatozoa to be performed; hence, severe oligozoospermic men were not included in the study. Although our cohort represents the largest one in the literature, the relatively small number of patients does not allow us to establish precisely the frequency of SDD at T2 which in our study reached 11-13% of patients. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide further insights into the long-term effects of cytotoxic treatments on the sperm genome. The persistent severe DNA damage after 2 years post-treatment observed in some patients suggests that there is an interindividual variation in restoring DNA integrity. We propose the use of SDF as a biomarker to monitor the treatment-induced genotoxic effects on sperm DNA in order to better personalize pre-conceptional counseling on whether to use fresh or cryopreserved spermatozoa. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Toscano Tumori (ITT), Fondazione Ente Cassa di Risparmio di Firenze, the European Commission-Reproductive Biology Early Research Training (REPROTRAIN). C.K., G.F., V.R., and A.R.-E. belong to COST Action CA20119 (ANDRONET) which is supported by the European Cooperation in Science and Technology (www.cost.eu). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.

BACKGROUND: Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number. OBJECTIVES: In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n = 39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n = 16). MATERIALS AND METHODS: We used a Vapour Fast Freezing procedure using 10 µL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n = 22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500 µL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test. RESULTS: We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n = 22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500 µL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500 µL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p < 0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p < 0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p < 0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p < 0.001). DISCUSSION AND CONCLUSION: Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.

Testicular and Haematological Cancer Induce Very High Levels of Sperm Oxidative Stress.

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.

Long-term effect of cytotoxic treatments on sperm DNA fragmentation in patients affected by testicular germ cell tumor.

INTRODUCTION: Testicular germ cell tumor is the most frequent neoplasia in men of reproductive age, with a 5-year survival rate of 95%. Antineoplastic treatments induce sperm DNA fragmentation, especially within the first year post-therapy. Data in the literature are heterogeneous concerning longer follow-up periods, and the large majority is limited to 2 years. OBJECTIVE: To define the timing for the recovery of sperm DNA damage and the proportion of patients with severe DNA damage at 2 and 3 years from the end of therapy. MATERIALS AND METHODS: Sperm DNA fragmentation was evaluated in 115 testicular germ cell tumor patients using terminal deoxynucleotidyl transferase dUTP nick end labeling assay coupled with flow cytometry before (T0 ) and 2 (T2 ) and 3 (T3 ) years post-treatment. Patients were divided based on the type of treatment: carboplatin, bleomycin-etoposide-cisplatin, and radiotherapy. For 24 patients, paired sperm DNA fragmentation data were available at all time-points (T0 -T2 -T3 ). Seventy-nine cancer-free, fertile normozoospermic men served as controls. Severe DNA damage was defined as the 95th percentile in controls (sperm DNA fragmentation = 50%). RESULTS: Comparing patients versus controls, we observed: (i) no differences at T0 and T3 and (ii) significantly higher sperm DNA fragmentation levels (p < 0.05) at T2 in all treatment groups. Comparing pre- and post-therapy in the 115 patients, the median sperm DNA fragmentation values were higher in all groups at T2 , reaching significance (p < 0.05) only in the carboplatin group. While the median sperm DNA fragmentation values were also higher in the strictly paired cohort at T2 , about 50% of patients returned to baseline. The proportion of severe DNA damage in the entire cohort was 23.4% and 4.8% of patients at T2 and T3 , respectively. DISCUSSION: Currently, testicular germ cell tumor patients are advised to wait 2 years post-therapy before seeking natural pregnancy. Our results suggest that this period may not be sufficient for all patients. CONCLUSION: The analysis of sperm DNA fragmentation may represent a useful biomarker for pre-conception counseling following cancer treatment.

Decrease of air pollution during lockdown in Tuscany (Italy): An effect on sperm DNA fragmentation?

In March 2020, the Italian government imposed a national lockdown which was almost completely removed in June 2020. Due to the abrupt stop of human activities, emissions of air pollutants decreased. Air pollution is an environmental risk factor for noncommunicable disease and mortality. Emerging evidence also suggests a role in male infertility. In this study, we compared sperm DNA fragmentation (sDF) levels and conventional semen parameters between subjects undergoing sDF determination and routine semen analysis in a single Italian centre, during about 6 months before (N = 119) and after lockdown (N = 105). After lockdown, we found an improvement of sperm progressive motility (48.00[38.50-58.00]% vs. 42.00[33.00-53.00]%) and sDF levels (as total: 24.79[18.33-33.97]% vs. 35.02[25.04-45.73]%, p < .001; brighter: 14.02[10.69-17.93]% vs 18.54[13.58-25.82]%, p < .001 and dimmer sDF: 9.24[5.64-15.78]% vs. 12.24[8.08-19.10]%, p < .01), mirrored by a decrease of leukocyte semen concentration (p < .01). The improvement of sperm motility and DNA quality was maintained after adjusting for leukocyte concentration and several conditions known to affect sperm motility and/or sDF levels. With a significant decrease in air pollution observed in Tuscany during and after lockdown, associated improvement in sperm motility and DNA quality in patients referred to the infertility clinic is suggestive of the potential role of air pollution in male infertility.

Vapour fast freezing with low semen volumes can highly improve motility and viability or DNA quality of cryopreserved human spermatozoa.

OBJECTIVES: To challenge a vapour fast freezing (VFF) cryopreservation procedure (conventional VFF) with several vitrification protocols and VFF conducted with small semen volumes (10 µl, microVFF), in order to implement a procedure for sperm banking in subjects with small sperm number. MATERIALS AND METHODS: Conventional VFF was conducted with test yolk buffer (TYB) as freezing medium and 500 µl straws as carriers. MicroVFF was conducted with TYB and using tips or cell sleepers as carriers. Vitrification was performed with TYB or SpermFreeze as freezing medium and with microspheres and tips as carriers. The effect of different procedures on progressive and total motility, viability, oxidative stress and DNA fragmentation of spermatozoa (sDF) was determined. Fresh and thawed samples, the latter after adequate washing/centrifuging, were evaluated. In some experiments, motility and viability recovery was determined in thawed samples, omitting the washing/centrifuging step. RESULTS: All the cryopreservation procedures blunted sperm motility and viability and induced increase of oxidative stress and sDF. However, VFF better preserved sperm motility and viability and less induced oxidative stress and sDF than vitrification, independently from the freezing medium and the carriers used in the latter. MicroVFF with cell sleepers resulted in a percentage increase of 57.58 ± 63.63%, 48.82 ± 74.96% and 24.55 ± 39.20% of, respectively, progressive and total motility and viability compared to the conventional VFF. Further, when tips were used, microVFF resulted in a percentage decrease of 15.77 ± 20.77% of sDF with respect to conventional VFF. Finally, omission of washing/centrifuging in post thawed samples, resulted in a much lower negative effect on motility and viability. DISCUSSION AND CONCLUSION: VFF, and in particular microVFF, better prevents sperm cryodamage than vitrification. Washing/centrifuging step after sample thawing seems to be responsible for a relevant fraction of damage to sperm motility and viability. Overall, our results are promising for developing a novel strategy of sperm banking in subjects with small sperm number, where low semen volumes are mandatory.

Novel methods to detect ROS in viable spermatozoa of native semen samples.

A crucial issue in male infertility work-up is to have reliable methods to detect oxidative stress in native semen samples. Here, we explored flow cytometric detection of Reactive Oxygen Species (ROS) in viable spermatozoa using native semen samples. To this aim, we challenged three fluorescent probes: CM-H2DCFDA, CellROX Green and MitoSOX Red. After excluding all non-sperm cells, each probe was coupled to a suitable stain to eliminate also semen apoptotic bodies and non-viable spermatozoa: Merocyanine 540 (M540) for CM-H2DCFDA and CellROX Green, and LIVE/DEAD Fixable Green Dead Cell Stain (LD-G) for MitoSOX Red. We found that CM-H2DCFDA was confined in the sperm midpiece, whereas CellROX Green and MitoSOX Red were localized in the head of spermatozoa. Treatment with H2O2 highly increased MitoSOX Red fluorescence (36.20 ± 5.24 vs 18.02 ± 2.25, %, p < 0.01), but not, or only slightly, the labelling with CMH2DCFDA (2.57 ± 1.70 vs 2.77 ± 1.43, p > 0.05) and CellROX Green (5.34 ± 3.18 vs 3.76 ± 2.04, p < 0.05), respectively. Menadione treatment highly increased CellROX Green (10.13 ± 5.85 vs 3.82 ± 2.70, p < 0.01) and MitoSOX Red (69.20 ± 27.14 vs 21.18 ± 7.96, %, p < 0.05), but not CM-H2DCFDA fluorescence (8.30 ± 11.56 vs 7.30 ± 9.19, p > 0.05). Further, only MitoSOX Red was able to detect spontaneous ROS generation during in vitro sperm incubation. We also detected DNA fragmentation by Comet and SCD Assay after sorting MitoSOX Red positive and negative sperm viable fractions. Results indicated that MitoSOX labelling in viable spermatozoa was strictly associated to sperm DNA fragmentation. In conclusion, MitoSOX Red/LD-G appears to be a promising method to detect oxidative stress in human semen for male infertility work-up.

Progesterone, spermatozoa and reproduction: An updated review.

The rapid effects of steroids on spermatozoa have been demonstrated for the first time more than three decades ago. Progesterone (P), which is present throughout the female genital tract with peaks of levels in the cumulus matrix surrounding the oocyte, has been shown to stimulate several sperm functions in vitro, including capacitation, hyperactivation, chemotaxis and acrosome reaction (AR). Besides an increase of intracellular calcium, P has been shown to activate other sperm signalling pathways including tyrosine phosphorylation of several sperm proteins. All these effects are mediated by extra-nuclear pathways likely involving interaction with molecules present on the sperm surface. In particular, the increase in intracellular calcium ([Ca2+]i) in spermatozoa from human and several other mammalian species is mediated by the sperm specific calcium channel CatSper, whose expression and function are required for sperm hyperactive motility. P-mediated CatSper activation is indeed involved in promoting sperm hyperactivation, but the involvement of this channel in other P-stimulated sperm functions, such as AR and chemotaxis, is less clear and further studies are required to disclose all the involved pathways. In human spermatozoa, responsiveness to P in terms of [Ca2+]i increase and AR is highly related to sperm fertilizing ability in vitro, suggesting that the steroid is a physiological inducer of AR during in vitro fertilization. In view of their physiological relevance, P-stimulated sperm functions are currently investigated to develop new tools to select highly performant spermatozoa for assisted reproduction.

DNA Fragmentation in Viable and Non-Viable Spermatozoa Discriminates Fertile and Subfertile Subjects with Similar Accuracy.

Sperm DNA fragmentation (sDF) negatively affects reproduction and is traditionally detected in total sperm population including viable and non-viable spermatozoa. Here, we aimed at exploring the ability of DNA fragmentation to discriminate fertile and subfertile men when detected in viable (viable sDF), non-viable (non-viable sDF), and total spermatozoa (total sDF). We revealed sDF in 91 male partners of infertile couples and 71 fertile men (max 1 year from natural conception) with LiveTUNEL coupled to flow cytometry, able to reveal simultaneously DNA fragmentation and cell viability. We found that the three sDF parameters discriminated fertile and subfertile men with similar accuracy and independently from age and basal semen parameters: AUCs (area under the curves) (95% CI) were: 0.696 (0.615-0.776), p < 0.001 for total sDF; 0.718 (0.640-0.797), p < 0.001 for viable sDF; 0.760 (0.685-0.835), p < 0.001 for non-viable sDF. We also found that total and non-viable but not viable sDF significantly correlated to age and semen quality. In conclusion, the three sDF parameters similarly discriminated fertile and subfertile men. Viable spermatozoa with DNA fragmentation are likely cells able to fertilize the oocyte but failing to properly support subsequent embryo development. Non-viable sDF could be a sign of a subtler damage extended beyond the non-viable cells.

Adverse effects of in vitro manipulation of spermatozoa.

Development of in vitro reproduction techniques has not only offered some infertile couples the possibility to have a child, it also revolutionized animal reproduction. Although in vitro reproduction techniques for humans or domestic and non-domestic animals have been designed to mimic in vivo conditions, modifications due to environmental effects or in vitro manipulation of gametes and embryos are unavoidable. For male gametes, in vitro manipulations include techniques to select spermatozoa, cryopreservation and other incubation procedures, during which spermatozoa may be exposed to oxidative stress and other insults that may damage their functions and DNA. The aim of this review is to provide an overview of key studies reporting sperm damage during in vitro manipulation, with particular focus on effects on DNA integrity, a fundamental factor for fertilization and transmission of paternal genetic information to offspring.

Teratozoospermia: Its association with sperm DNA defects, apoptotic alterations, and oxidative stress.

This study was carried out to evaluate the level of nuclear sperm DNA damage in men with isolated polymorphic teratozoospermia and examining its relationship with apoptosis and oxidative stress. A total of 89 subjects were divided into two groups: men with isolated teratozoospermia (n = 69) and men with normal semen parameters (n = 20) as controls. Sperm DNA breaks were determined by using acridine orange staining. The proportion of viable spermatozoa with mitochondrial transmembrane depolarization was detected by fluorescence microscopy through the use of MitoPTJC-1 staining method. Bivariate Annexin V/6-CFDA analysis was then set out in order to measure the percentage of both viable and dead spermatozoa with phosphatidylserine (PS) externalization. Seminal antioxidant profile (reduced glutathione (GSHr); oxidized glutathione (GSSG); glutathione S-transferase (GST)) and total protein sulfhydryl (P-SH) concentrations were measured spectrophotometrically. Men with isolated teratozoospermia, when compared to controls, showed significantly increased level of single sperm DNA breaks and higher proportions of spermatozoa with phosphatidylserine externalization and mitochondrial depolarization. Among the differently studied oxidative stress seminal parameters, the rates of seminal GSHr, GST, and P-SH were significantly decreased in the teratozoospermic group. However, the seminal rates of GSSG and GST have decreased, but only GST did not show a significant difference. Interestingly, significant correlations were found between the studied apoptotic markers and the rate of atypical sperm forms with the incidences of head abnormalities. Furthermore, positive inter-correlations were found between sperm DNA defects, impaired seminal antioxidant status, and the apoptotic sperm markers. Such data provide clear evidence that the apoptotic alterations are closely correlated to abnormal sperm morphology and DNA damage. Moreover, decreased seminal antioxidant profile may be a crucial factor involved in the mechanism of sperm cell death-mediated DNA breaks in teratozoospermic semen.

Sperm DNA Fragmentation: Mechanisms of Origin.

Spermatozoa have the task to deliver an intact paternal genome to the oocyte and to support a successful embryo development. The high levels of sperm DNA fragmentation (sDF) found in sub-/infertile men threat human reproduction and health of the offspring. Strategies to prevent the onset of this type of sperm damage are extensively sought.sDF can be induced by factors like lifestyle-related habits, diseases, drugs, aging, infections and exposure to pollutants. At the cell level, all these factors induce sperm DNA breaks by three main mechanisms: apoptosis, impairment of sperm chromatin maturation and oxidative stress. Apoptosis and defects in maturation of sperm chromatin appear to act in the testis and account for DNA breaks found in dead ejaculated spermatozoa, whereas oxidative stress is likely inducing sDF during the transit through the male genital tracts and accounting for DNA breaks observed in viable spermatozoa of the ejaculate. Oxidative stress appears to be also the main mechanism responsible for induction of sDF after ejaculation, during in vitro manipulation of spermatozoa. Whether or not mature spermatozoa are able to trigger a cell death program is not yet clarified. In particular, it is not clear whether apoptotic nucleases or reactive oxygen species are responsible for producing DNA breaks in ejaculated mature spermatozoa. Knowledge of the mechanisms inducing sDF is a valuable starting point to define possible therapeutic options that however are still far to be established.

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility.

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Chromatin condensation, fragmentation of DNA and differences in the epigenetic signature of infertile men.

Epidemiological studies report an increase of pathologies of male reproductive tracts and suggest a link between this trend and the increased exposure of men to endocrine disruptors (EDs). The mechanisms by which EDs impact male fertility are far to be elucidated although DNA, chromatin and epigenome of spermatozoa appear to be relevant targets for these molecules. Indeed, many studies report associations between increased levels of sperm DNA fragmentation (sDF) or aberrant chromatin condensation or epigenetic modifications and poor semen quality and/or infertile phenotype. In this scenario, therapies able to reduce sperm damage to DNA, chromatin and epigenome are sought. Currently, antioxidants and FSH administration is proposed for treating high levels of sDF, but whether or not such therapies are really effective is still debated. Further studies are necessary to understand the link between endocrine disruptor exposure and damage to sperm function and/or structure and thus to define effective therapeutic strategies.

Effects of FSH on Sperm DNA Fragmentation: Review of Clinical Studies and Possible Mechanisms of Action.

Sperm DNA fragmentation (sDF) is an important reproductive problem, associated to an increased time-to-pregnancy and a reduced success rate in natural and in vitro fertilization. sDF may virtually originate at any time of sperm's life: in the testis, in the epididymis, during transit in the ejaculatory ducts and even following ejaculation. Studies demonstrate that an apoptotic pathway, mainly occurring in the testis, and oxidative stress, likely acting in the male genital tract, are responsible for provoking the DNA strand breaks present in ejaculated spermatozoa. Although several pharmacological anti-oxidants tools have been used to reduce sDF, the efficacy of this type of therapies is questioned. Clearly, anti-apoptotic agents cannot be used because of the ubiquitous role of the apoptotic process in the body. A notable exception is represented by Follicle-stimulating hormone (FSH), which regulates testis development and function and has been demonstrated to exert anti-apoptotic actions on germ cells. Here, we review the existing clinical studies evaluating the effect of FSH administration on sDF and discuss the possible mechanisms through which the hormone may reduce sDF levels in infertile subjects. Although there is evidence for a beneficial effect of the hormone on sDF, further studies with clear and univocal patient inclusion criteria, including sDF cut-off levels and considering the use of a pharmacogenetic approach for patients selection are warranted to draw firm conclusions.

Massive Weight Loss Obtained by Bariatric Surgery Affects Semen Quality in Morbid Male Obesity: a Preliminary Prospective Double-Armed Study.

OBJECTIVES: The aim of this study is to evaluate the effect of massive weight loss on the seminal parameters at 6 months from bariatric surgery. DESIGN: Two-armed prospective study performed in 31 morbidly obese men, undergoing laparoscopic roux-en-Y-gastric bypass (n = 23) or non-operated (n = 8), assessing sex hormones, conventional (sperm motility, morphology, number, semen volume), and non-conventional (DNA fragmentation and seminal interleukin-8), semen parameters, at baseline and after 6 months from surgery or patients' recruitment. RESULTS: In operated patients only, a statistically significant improvement in the sex hormones was confirmed. Similarly, a positive trend in the progressive/total sperm motility and number was observed, though only the increase in semen volume and viability was statistically significant (Δ = 0.6 ml and 10%, P < 0.05, respectively). A decrease in the seminal interleukin-8 levels and in the sperm DNA fragmentation was also present after bariatric surgery, whereas these parameters even increased in non-operated subjects. Age-adjusted multivariate analysis showed that the BMI variations significantly correlated with the changes in the sperm morphology (ß = -0.675, P = 0.025), sperm number (ß = 0.891, P = 0.000), and semen volume (r = 0.618, P = 0.015). CONCLUSION: The massive weight loss obtained with bariatric surgery was associated with an improvement in some semen parameters. The correlations found between weight loss and semen parameter variations after surgery suggest that these might occur early downstream of the testis and more slowly than the changes in the sex hormones.

TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation.

Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.

Sperm DNA fragmentation in cryopreserved samples from subjects with different cancers.

Sperm cryopreservation is widely used by cancer patients undergoing chemo- or radiotherapy. Evidence suggests that IVF outcome with cryopreserved spermatozoa from cancer patients is less successful. To determine whether sperm DNA fragmentation (SDF) is involved in the lower fertilising ability of cryopreserved spermatozoa of cancer patients, SDF was evaluated in thawed spermatozoa from 78 men affected by different cancers and 53 men with non-cancer pathologies. SDF was assessed by the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), propidium iodide (PI), flow cytometry procedure, which allows determination of two different cell populations (PIbrighter and PIdimmer) and thus to determine the percentage of DNA fragmented sperm in both. PIdimmer spermatozoa are totally unviable, whereas PIbrighter spermatozoa with SDF may be motile and morphologically normal, having higher biological relevance in the reproductive process. We found that the proportion of DNA fragmented PIbrighter cells was significantly higher in thawed spermatozoa from cancer than non-cancer patients. Moreover, a positive correlation was found between the degree of DNA fragmentation and sperm motility in the PIbrighter population of spermatozoa from cancer patients that wasn't seen in non-cancer patients. The results of the present study suggest that higher SDF levels may contribute to the lower IVF success of cryopreserved spermatozoa from cancer patients and that evaluation of SDF could complement genetic counselling as part of the routine management of cancer patients who seek fertility preservation.

DNA fragmentation in two cytometric sperm populations: relationship with clinical and ultrasound characteristics of the male genital tract.

We investigated whether DNA fragmentation in two cytometric sperm populations (PIdimmer and PIbrighter) with different biological characteristics and clinical relevance is related to clinical and color-Doppler ultrasound (CDUS) parameters of the male genital tract. One hundred and sixty males of infertile couples without genetic abnormalities were evaluated for clinical, scrotal, and transrectal CDUS characteristics, presence of prostatitis-like symptoms (with the National Institutes of Health-Chronic Prostatitis Symptom Index) and sperm DNA fragmentation (sDF) in PIdimmer and PIbrighter populations (using TUNEL/PI method coupled with flow cytometry). Data were adjusted for age (Model 1) along with waistline, testosterone levels, smoking habit, and sexual abstinence (Model 2). According to the statistical Model 2, PIdimmer sDF was associated with testicular abnormalities, including lower clinical and ultrasound volume (r = -0.21 and r = -0.20, respectively; P < 0.05), higher FSH levels (r = 0.34, P < 0.0001) and occurrence of testicular inhomogeneity (P < 0.05) and hypoechogenicity (P < 0.05). PIbrighter sDF was associated with prostate-related symptoms and abnormal signs, including higher NIH-CPSI total and subdomain scores, a higher prevalence of prostatitis-like symptoms and of CDUS alterations such as macro-calcifications, severe echo-texture inhomogeneity, hyperemia (all P < 0.05), and higher arterial peak systolic velocity (r = 0.25, P < 0.05). Our results suggest that DNA fragmentation in PIdimmer sperm, which is related to poor semen quality, mainly originates in the testicles, likely due to apoptosis. Conversely, DNA fragmentation in PIbrighter sperm appears to mainly originate during or after transit through the prostate, increasing with the presence of an inflammatory status of the organ. These results could lead to new perspectives for the identification of therapeutic targets to reduce sDF.