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.

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.

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.

Treatment with human, recombinant FSH improves sperm DNA fragmentation in idiopathic infertile men depending on the FSH receptor polymorphism p.N680S: a pharmacogenetic study.

STUDY QUESTION: Does the sperm DNA fragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? SUMMARY ANSWER: FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. WHAT IS KNOWN ALREADY: FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. STUDY DESIGN, SIZE AND DURATION: A multicenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygous N or S genotype, FSH ≤ 8 IU/l and DFI >15%, were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty-eight men (57.6%) were carriers of the p.N680S homozygous N and 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680S N versus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFI was inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014). When patients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G>T polymorphism in the FSHB gene (rs10835638) (homozygous G versus others), the significant improvement of sperm DFI in FSHR p.N680S homozygous N men was independent of sperm concentration and associated with the homozygous FSHB -211G>T homozygous G genotype. LIMITATIONS, REASONS FOR CAUTION: The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygous N FSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. WIDER IMPLICATIONS OF THE FINDINGS: The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. STUDY FUNDING/COMPETING INTERESTS: The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. TRIAL REGISTRATION NUMBER: EudraCT number 2010-020240-35.

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress.

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2'-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.

The CatSper calcium channel in human sperm: relation with motility and involvement in progesterone-induced acrosome reaction.

STUDY QUESTION: Does CatSper have a role in the achievement of human sperm motility and in the Progesterone (P)-induced acrosome reaction (AR)? SUMMARY ANSWER: CatSper1 expression is associated with human sperm progressive motility and the P-induced AR; it may have a role in the pathogenesis of asthenozoospermia. WHAT IS KNOWN ALREADY: Knockout mice for any of the Catsper family genes fail to acquire hyperactivated motility and are infertile. CatSper channels mediate P-induced Ca(2+) influx in human sperm. The role of CatSper in human sperm hyperactivated/activated motility and in asthenospermia is less clear. A few men with CatSper mutations have been described but the phenotype regarding sperm motility has not been well established. STUDY DESIGN, SIZE, DURATION: The effects of two Catsper inhibitors, NNC55-0396 (NNC, 10 and 20 µM) and Mibefradil (Mib, 30 and 40 µM), were tested on human sperm motility parameters and the P-induced AR. Catsper1 protein expression was evaluated in unselected and swim-up selected sperm samples and in sperm from normo- and astheno-zoospermic subjects. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen sample kinematic parameters were analysed by a CASA system. A fluorescent-labelled lectin was used to evaluate P-induced AR in live sperm by fluorescence microscopy. CatSper1 protein expression was determined by western blot analysis and by flow cytometry. Intracellular calcium concentrations ([Ca(2+)]i) were evaluated by a spectrofluorimetric method following sperm loading with the calcium-sensitive probe fura 2/AM. MAIN RESULTS AND THE ROLE OF CHANCE: CatSper1 protein was localized in the tail of human sperm. CatSperI expression was higher in swim up selected than unselected sperm both when measured by western blot or by flow cytometry (52.7 ± 15.8% versus 27.2 ± 9.01%, n = 7, P < 0.01). Basal and P-stimulated [Ca(2+)]i were significantly higher in swim-up selected compared with unselected sperm. CatSper1 expression (western blot analysis) was found to be decreased in sperm from asthenozoospermic (n = 10) compared with those from normozoospermic (n = 9) men (intensity values relative to ß-actin: 244.4 ± 69.3 versus 385.8 ± 139.5, P < 0.01). A positive correlation was found between CatSper1 protein expression and the percentage of sperm with progressive motility (n = 19, r = 0.59, P = 0.007). NNC (10 µM) and Mib (30 µM) significantly reduced the percentage of sperm with progressive motility and several kinematic parameters but did not affect the percentage of hyperactivated sperm. Their effects were the same whether they were added to swim-up selected and capacitated sperm or were added to the swim-up medium. Mib was found to have a slight but significant effect on sperm viability at both concentrations tested. P-stimulated AR was significantly reduced by both inhibitors (P < 0.05). Overall, our results indicate that, in human sperm, CatSper channel expression and function are associated with progressive motility and may be involved in the pathogenesis of asthenozoospermia. LIMITATIONS, REASONS FOR CAUTION: In general, studies evaluating the effect of inhibitors have the limitation of the specificity of the molecules. We show here that Mib may have toxic effect on human sperm. Although most of the parameters have been evaluated in live sperm, the toxic effect could have contributed to the observed decreases. More studies are necessary to evaluate further the role of CatSper1 in asthenozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: In view of the involvement in P-induced AR and of the evidence of a role in the pathogenesis of astenozoospermia, CatSper channels may represent a promising target for the development of new drugs for the treatment of male infertility and for non-hormonal contraception. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Ministry of University and Scientific Research (Prin project to E.B. and FIRB project to S.M) and Regione Toscana (to G.F.). The authors have no conflicts of interest to declare.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular markers of human sperm functions.

Fertilization is a stepwise process that allows two mature gametes to reach each other, fuse and eventually give rise to a new individual. Despite the tremendous importance of reproduction for species development and maintenance, fertility is decreasing worldwide, with peaks in western countries. It is estimated that about 7% of men experiences problems in conceiving a child because of sperm defects. In such a situation, understanding which are the essential sperm players in each of the steps of the fertilization process is essential for the development of new pharmacological strategies to treat the infertile men, for genetic screening of idiopathic male infertility as well as to produce effective male contraceptive agents. The present review will summarize recent evidence for the identification and characterization of molecular markers of sperm functions with emphasis on post-ejaculatory maturation events and the process of sperm-oocyte interaction.

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.

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.

Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labeling.

Conflicting results are reported by recent studies comparing flow cytometry (FCM) and fluorescence microscopy (FM) for detecting sperm DNA fragmentation by TUNEL assay. Each of the two technologies has specific peculiarities and limitations, but whereas the limitations of FM observation are well known, the biases due to the inability of FCM to recognize morphologically analyzed cells are less explored. In particular, so far, FCM analysis of sperm DNA fragmentation have included in the analyses M540 bodies, round semen structures exhibiting FSC/SSC properties similar to sperm. Semen M540 bodies, altogether with the occurrence of two sperm populations with different nuclear staining, concur to an underestimation of values of sperm DNA fragmentation by FCM. However, even considering such bias, the observed discrepancies between the performance of FM and FCM are not fully explained. We discuss here the possible variables that may affect the results of each of the two technologies and the necessary efforts to be taken to address this issue.