Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

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.

Semen Cryopreservation for Men Banking for Oligozoospermia, Cancers, and Other Conditions: 24 Years' Experience of an Italian Bank.

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.

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.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: An overview on male genital tract ultrasound reference ranges.

BACKGROUND: So far, male genital tract color-Doppler ultrasound (MGT-CDUS) was not standardized. Recently, the European Academy of Andrology (EAA) published the results of a multicenter study assessing the CDUS characteristics of healthy-fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report the EAA US study (i) standard operating procedures (SOPs) for assessing MGT-CDUS, (ii) main MGT-CDUS normative parameters, and (iii) compare the EAA and previously published "normal" CDUS values. METHODS: A cohort of 248 HFM (35.3 ± 5.9 years) was studied, evaluating MGT-CDUS before and after ejaculation following SOPs. RESULTS: SOPs for MGT-CDUS assessment are summarized here. All subjects underwent scrotal CDUS and 188 men underwent transrectal ultrasound before and after ejaculation. The main CDUS reference ranges and characteristics of the HFM-MGT are reported here. The mean testicular volume was ∼17 mL. The lower limit for right and left testis was 12 and 11 mL, defining testicular hypotrophy. The upper limit for epididymal head, body, tail, and vas deferens was 11.5, 5, 6, and 4.5 mm, respectively. Testicular and epididymal arterial reference ranges are reported. The EAA varicocoele classification is reported. CDUS-varicocoele was detected in ∼37% of men. Prostate mean volume was ∼25 mL, while lower and upper limits were 15 and 35 mL, defining hypotrophy and enlargement, respectively. Prostate arterial reference ranges are reported. Prostate calcifications and inhomogeneity were frequent; midline prostatic cysts were rare and small. Ejaculatory duct abnormalities were absent. The upper limit for periprostatic venous plexus was 4.5 mm. Lower and upper limits of seminal vesicles (SV) anterior-posterior diameter were 6 and 16 mm, defining hypotrophy or dilation, respectively. Seminal vesicle volume and ejection fraction reference ranges are reported. SV-US abnormalities were rare. Deferential ampullas upper limit was 6 mm. A discussion on the EAA and previously published "normal" CDUS values is reported here. CONCLUSIONS: The EAA findings will help in reproductive and general male health management.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Prostate-vesicular transrectal ultrasound reference ranges and associations with clinical, seminal and biochemical characteristics.

BACKGROUND: Transrectal ultrasound (TRUS) parameters are not standardized, especially in men of reproductive age. Hence, the European Academy of Andrology (EAA) promoted a multicenter study to assess the TRUS characteristics of healthy-fertile men (HFM) to establish normative parameters. OBJECTIVES: To report and discuss the prostate and seminal vesicles (SV) reference ranges and characteristics in HFM and their associations with clinical, seminal, biochemical parameters. METHODS: 188 men (35.6 ± 6.0 years) from a cohort of 248 HFM were studied, evaluating, on the same day, clinical, biochemical, seminal, TRUS parameters following Standard Operating Procedures. RESULTS: TRUS reference ranges and characteristics of the prostate and SV of HFM are reported herein. The mean PV was ∼25 ml. PV lower and upper limits were 15 and 35 ml, defining prostate hypotrophy and enlargement, respectively. PV was positively associated with age, waistline, current smoking (but not with T levels), seminal volume (and negatively with seminal pH), prostate inhomogeneity, macrocalcifications, calcification size and prostate arterial parameters, SV volume before and after ejaculation, deferential and epididymal size. Prostate calcifications and inhomogeneity were frequent, while midline prostatic cysts were rare and small. Ejaculatory duct abnormalities were absent. Periprostatic venous plexus size was positively associated with prostate calcifications, SV volume and arterial peak systolic velocity. Lower and upper limits of SV anterior-posterior diameter after ejaculation were 6 and 16 mm, defining SV hypotrophy or dilation, respectively. SV total volume before ejaculation and delta SV total volume (DSTV) positively correlated with ejaculate volume, and DSTV correlated positively with sperm progressive motility. SV total volume after ejaculation was associated negatively with SV ejection fraction and positively with distal ampullas size. SV US abnormalities were rare. No association between TRUS and time to pregnancy, number of children or history of miscarriage was observed. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology and the meaning of specific TRUS findings.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: clinical, seminal and biochemical characteristics.

BACKGROUND: Infertility affects 7%-12% of men, and its etiology is unknown in half of cases. To fill this gap, use of the male genital tract color-Doppler ultrasound (MGT-CDUS) has progressively expanded. However, MGT-CDUS still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study ("EAA ultrasound study") to assess MGT-CDUS characteristics of healthy, fertile men to obtain normative parameters. OBJECTIVES: To report (a) the development and methodology of the "EAA ultrasound study," (b) the clinical characteristics of the cohort of healthy, fertile men, and (c) the correlations of both fertility history and seminal features with clinical parameters. METHODS: A cohort of 248 healthy, fertile men (35.3 ± 5.9 years) was studied. All subjects were asked to undergo, within the same day, clinical, biochemical, and seminal evaluation and MGT-CDUS before and after ejaculation. RESULTS: The clinical, seminal, and biochemical characteristics of the cohort have been reported here. The seminal characteristics were consistent with those reported by the WHO (2010) for the 50th and 5th centiles for fertile men. Normozoospermia was observed in 79.6% of men, while normal sperm vitality was present in almost the entire sample. Time to pregnancy (TTP) was 3.0[1.0-6.0] months. TTP was negatively correlated with sperm vitality (Adj.r =-.310, P = .011), but not with other seminal, clinical, or biochemical parameters. Sperm vitality and normal morphology were positively associated with fT3 and fT4 levels, respectively (Adj.r = .244, P < .05 and Adj.r = .232, P = .002). Sperm concentration and total count were negatively associated with FSH levels and positively, along with progressive motility, with mean testis volume (TV). Mean TV was 20.4 ± 4.0 mL, and the lower reference values for right and left testes were 15.0 and 14.0 mL. Mean TV was negatively associated with gonadotropin levels and pulse pressure. Varicocoele was found in 33% of men. CONCLUSIONS: The cohort studied confirms the WHO data for all semen parameters and represents a reference with which to assess MGT-CDUS normative parameters.

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.

Seminal but not Serum Levels of Holotranscobalamin are Altered in Morbid Obesity and Correlate with Semen Quality: A Pilot Single Centre Study.

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility (r = 0.394, p = 0.012), sperm concentration (r = 0.401, p = 0.009), total sperm number (r = 0.343, p = 0.028), and negative correlation with semen pH (r = -0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

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.

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.

Acrosome reaction is impaired in spermatozoa of obese men: a preliminary study.

OBJECTIVE: To compare spontaneous (Sp-AR) and P-induced acrosome reaction (AR) in spermatozoa of obese and lean subjects. SETTING: Bariatric unit at a university hospital. DESIGN: Prospective, observational study. PATIENT(S): Twenty-three obese (mean±SD body mass index [BMI], 44.3±5.9 kg/m2) and 25 age-matched lean (BMI, 24.2±3.0 kg/m2) subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Spontaneous and P-induced AR in spermatozoa of obese and lean subjects. RESULT(S): A statistically significant difference was found between obese and lean cohorts in total T and calculated free T, E2, glycated hemoglobin, and high-density lipoproteins, whereas among the routine semen parameters analyzed, only immotile sperm percentage and ejaculate volume differed significantly. Spermatozoa of obese (n=13) vs. lean men (n=19) showed a higher Sp-AR (17.9%±7.2% vs. 8.3%±4.2%), which resulted in a reduced ability to respond to P evaluated as the AR-after-P-challenge parameter (3.5%±3.2% vs. 17.6%±9.2%). Multivariate analysis adjusted for age revealed a significant correlation between BMI, waist, E2, and glycated hemoglobin with both Sp-AR (age-adjusted r=0.654, r=0.711, r=0.369, and r=0.644, respectively) and AR-after-P-challenge (age-adjusted r=-0.570, r=-0.635, r=-0.507, and r=-0.563, respectively). A significant difference in sperm cholesterol content was reported between obese and lean men (29.8±19.5 vs. 19.1±14.6 ng/µg of proteins). CONCLUSION(S): Sperm AR is impaired in obese men, showing reduced response to P and elevated Sp-AR, associated with altered circulating levels of E2 and sperm cholesterol content.

Semen cryopreservation for men banking for oligospermia, cancers, and other pathologies: prediction of post-thaw outcome using basal semen quality.

OBJECTIVE: To evaluate post-thawing sperm parameters in a large series of men cryopreserving for different cancers and oligospermia. DESIGN: Retrospective observational study. SETTING: Semen cryopreservation laboratory. PATIENT(S): Six hundred twenty-three patients undergoing semen cryopreservation for cancer or oligospermia who discontinued banking. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Postcryopreservation sperm motility and viability. RESULT(S): In oligospermic men, recovery of motile sperm after cryopreservation was possible in only a few out of the 219 samples cryopreserved for this problem. Similarly, independent of the reason for which cryopreservation was required, if one basal semen parameter fell below the 5th percentile of the World Health Organization reference values, recovery of motile and viable spermatozoa after thawing was low. Among samples cryopreserved for cancer, those with testicular cancer showed the lowest basal semen quality and recovery after thawing. In cases of hematological cancers or other types of cancers, motility recovery was similar to that of non-cancer-related samples. Receiver operating characteristic analyses demonstrate that basal progressive and total motility predict the recovery rate of motile sperm after thawing with high accuracy, sensibility and specificity. CONCLUSION(S): Our study demonstrates the ability of prefreeze semen parameters to predict cryosurvival in terms of sensitivity and precision. Using this information, the clinician could perform appropriate counseling about the future possibilities of fertility for the patient.

Peroxisome-proliferator-activated receptor gamma (PPARgamma) is required for modulating endothelial inflammatory response through a nongenomic mechanism.

Besides their well-known anti-diabetic effects, the peroxisome-proliferator-activated receptor gamma (PPARgamma) thiazolidinedione ligands (TZD) have been suggested to also display anti-inflammatory properties. The receptor role in mediating such effects is far from being elucidated. Here, we demonstrated that PPARgamma is necessary for TZD to interfere with TNFalpha and IFNgamma inflammatory activity in human endothelial cells. Different PPARgamma ligands similarly inhibit cytokinic stimulation of IFNgamma-inducible-protein-of-10-kDa (IP10) secretion in a dose-dependent manner and prevent the induced phosphorylation/activation of extracellular-signaling-regulated-kinases (ERK1/2). To further confirm the PPARgamma role in mediating both rapid and long term anti-inflammatory effects of its ligands, we evaluated RGZ inhibitory action in PPARgamma-silenced and -overexpressing cells. PPARgamma-silencing results in a reversion of RGZ inhibitory activity on cyto/chemokine secretions and rapid ERK phosphorylation. Conversely, receptor overexpression significantly increases RGZ inhibitory activity. Finally, PPARgamma-overexpression results in a reduction of ERK1/2 phosphorylation and inflammatory secretions in response to TNFalpha and IFNgamma even in the absence of RGZ, suggesting a restraining effect controlled by endogenous ligands. In conclusion, our data provide the first evidence that PPARgamma is involved in the anti-inflammatory action of TZD in endothelial cells, not only by modulating cyto/chemokine secretions but also by restraining ERK activation through a novel rapid nongenomic mechanism.

Molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor alpha and interferon gamma in human microvascular endothelium.

Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) are among the most potent cytokines involved in orchestrating the inflammation response. The molecular mechanisms implicated in the synergism between cytokines are still poorly characterized. We demonstrate that both cytokines dose-dependently stimulate IFNgamma-inducible-protein-of-10-kDa (IP-10) secretion in human microvascular endothelial cells (HMEC-1), showing a potent synergism which is not restricted to IP-10, but is also evident for monokine-induced-by-IFNgamma (MIG) and IL-6 secretion. Immunofluorescence analysis reveals that TNFalpha and IFNgamma converge on a rapid phosphorylation of ERK, which however results in a different subcellular compartmentalization of the activated enzyme in response to the two cytokines. Differences in the subcellular recruitment of ERK in response to IFNgamma and TNFalpha are responsible for generating different ERK downstream signaling, which can thus synergize on the secretion of IP-10 as well as of other cytokines/chemokines. The importance of ERK activation in mediating the synergism of the two cytokines is further confirmed by the inhibitory effect of the anti-diabetic drug rosiglitazone and ERK blockers on IP-10, MIG and IL-6 secretion. A further mechanism of synergism involving the reciprocal upregulation of TNFalpha-RII and of IFNgamma-R, in response to IFNgamma and TNFalpha, respectively, was revealed by flow cytometry and quantitative real time RT-PCR analysis.

Natural transmission of USP9Y gene mutations: a new perspective on the role of AZFa genes in male fertility.

Deletions of the azoospermia factor (AZF) regions of the Y chromosome are associated with severe spermatogenic failure and represent the most frequent molecular genetic cause of azoospermia and severe oligozoospermia. The exact role of the candidate AZF genes is largely unknown due to both the extreme rarity of naturally occurring AZF gene-specific mutations and the lack of functional assays. Here, we report the fine characterization of two different deletions in the USP9Y gene (one of the two candidate genes in the AZFa region), which have been transmitted through natural conception in two unrelated families. The associated mild testicular phenotype, in both cases, is in sharp contrast with that of the two previously reported infertile patients bearing a mutation of the same gene. In conclusion, to date, the USP9Y gene has been considered as one of the major Y-linked spermatogenesis genes, based on both its position within the AZFa region and previous reports that correlated USP9Y mutation to severe spermatogenic failure and infertility. This view is now substantially changed because our findings clearly demonstrate that during human spermatogenesis, USP9Y is more likely a fine tuner that improves efficiency, rather than a provider of an essential function. More importantly, the observed natural conceptions suggest that the protein is not required for the final sperm maturation process or for the acquisition of sperm fertilizing ability, providing a new perspective on the role played by the USP9Y gene in male fertility.

Y chromosome and male infertility: update, 2006.

Male factor infertility accounts for about half the cases of couple infertility and in around 50% of cases its etiology remains unknown. Molecular genetic techniques have unveiled a number of etiopathogenetic factors, including microdeletions of the Yq. Y chromosome microdeletions removing the AZoospermia Factor (AZF) regions are the most frequent molecular genetic causes of oligo/azoospermia. The intense effort of many laboratories contributed to a better understanding of the clinical significance of this genetic anomaly and to the identification of fertility candidate genes in the AZF regions. Important progress has been made on the structure of the Y chromosome and the mechanism of deletion. Studies aimed to define a predisposing genetic background for Yq deletions were not successful, perhaps due to the low number of patients analyzed so far. The screening for Yq deletions became a routine diagnostic test that provides an etiology for spermatogenic disturbances, and assess in the prognosis for testicular sperm retrieval according to the type of deletion. Assisted reproductive techniques represent an efficient symptomatic therapy for men bearing Y microdeletions, however, this genetic defect is transmitted to the male offsprings, affecting their fertility. Future studies should focus on understanding the biological function of AZF genes which is an essential step for the development of more appropriate and knowledge-based therapies.