Luca De Toni

High-power microscopy for selecting spermatozoa for ICSI by physiological status

Sperm selection for intracytoplasmic sperm injection (ICSI), based on standard morphology, can fail to select normal cells, and actual methods to evaluate their physiological status do not allow their later use for ICSI. Some authors have demonstrated that sperm selection based on high-magnification morphology is associated with a better ICSI outcome, above all in subjects with severe testicular failure. In this study there was an evaluation of mitochondrial function, chromatin structure and sperm aneuploidies on whole sperm samples from 30 subjects: 10 normozoospermic controls and 20 patients that were severely oligozoospermic due to testicular damage or partial obstruction of the seminal ducts. All severely oligozoospermic patients showed worse mitochondrial function and chromatin status, while sperm aneuploidies were significantly increased only in those subjects with severe testicular damage (P < 0.001). In the latter patients the analysis of a single spermatozoon, performed after morphological selection by high-magnification microscopy, showed significantly better mitochondrial function, chromatin status and aneuploidy rate than observed in unselected cells (all P < 0.001). Interestingly, these parameters were further improved when nuclear vacuoles were lacking. These results suggest a strong relationship between high-magnification morphology and the status of spermatozoa, and they may explain the better results of ICSI obtained using spermatozoa selected by high-magnification microscopy.

Evidence for osteocalcin production by adipose tissue and its role in human metabolism.

CONTEXT The adipose tissue (AT), which is an endocrine organ, is linked to several metabolic abnormalities. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion. OBJECTIVE Our objective was to investigate the involvement of OCN in obesity and to evaluate, in vitro and ex vivo, the role of AT in the modulation of this endocrine circuit. DESIGN, PATIENTS, AND SETTING This transversal study involved 83 male subjects, divided according to the World Health Organization body mass index classification, evaluated at Padovas Obesity Outpatient Clinic. METHODS OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA. OCN mRNA expression and protein production were measured by quantitative RT-PCR and immunohistochemistry during in vitro adipogenesis and in sc AT (SAT) and omental AT (OAT) from normal adult men. cOCN and ucOCN release by AT in a simple growth medium was verified by ELISA. RESULTS Overweight and obese patients had a lower ucOCN and ucOC/OCN ratio. In the whole cohort, ucOCN/OCN ratio was negatively correlated to body mass index (rho = -0.233; P < 0.05). OCN mRNA was present in SAT and OAT and during all stages of adipogenesis, with higher expression in the first steps. Immunohistochemistry confirmed the expression of OCN protein. Both SAT and OAT were able to release cOCN and ucOCN. CONCLUSIONS Our data support a pathophysiological link between ucOCN and cOCN balance and obesity. OCN is present in the first phases of adipogenesis but also in human AT ex vivo. AT releases, in vitro, both ucOCN and cOCN, suggesting a possible link between AT and OCN in the regulation of metabolism.

Androgens stimulate endothelial progenitor cells through an androgen receptor-mediated pathway

Background and objective  Testosterone (T) treatment has recently been shown to induce an increase in the number of endothelial progenitor cells (EPCs) through a possible effect on bone marrow. Hypogonadotrophic hypogonadal (HH) men have low circulating EPCs that increase significantly after T treatment. Moreover, expression of the androgen receptor (AR) has been demonstrated by immunohistochemistry in these cells, suggesting that T might also have a direct effect on EPC function. In the present study we investigated the expression and function of the AR in human EPCs and the in vitro effect of androgens on EPC function.

Bone Mineral Density and Testicular Failure: Evidence for a Role of Vitamin D 25-Hydroxylase in Human Testis

WORKING HYPOTHESIS Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets. OBJECTIVE Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients. DESIGN, PATIENTS, SETTING: Testis samples for in vitro study and 98 young men were transversally evaluated at Padovas Center for Male Gamete Cryopreservation. METHODS CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis. RESULTS We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05). CONCLUSIONS Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.

ORIGINAL RESEARCH—BASIC SCIENCE: The PDE5 Inhibitor Sildenafil Increases Circulating Endothelial Progenitor Cells and CXCR4 Expression

INTRODUCTION Endothelial progenitor cells (EPC) are a specific subtype of progenitor cells that can be isolated from circulating mononuclear cells, able to migrate from the bone marrow to the peripheral circulation where they contribute to vascular repair. CXC-motif chemochine receptor 4 (CXCR4) receptor seems to play a critical role in this process. AIM To assess the effects of sildenafil (a type 5 phosphodiesterase [PDE5] inhibitor) administration in 20 healthy young men. METHODS Evaluation of CXCR4 expression in circulating EPC before and 4 hours after in vivo administration of 100 mg sildenafil by flow cytometry and colony-forming unit. RESULTS We found that sildenafil increases circulating EPC number, the relative expression of CXCR4 on these cells and the ability to generate colonies in vitro. CONCLUSIONS These data allow us to suppose an involvement of PDE5 in bone marrow release and peripheral homing of EPC.

Human papillomavirus sperm infection and assisted reproduction: a dangerous hazard with a possible safe solution

BACKGROUND Human papillomavirus (HPV) infection has been demonstrated in the sperm of a large percentage of sexually active males and is associated with an impairment of sperm parameters, with a particular negative impact on sperm motility, suggesting a possible role in male infertility. Conventional sperm selection techniques have a low efficiency in removing HPV. METHODS Evaluation of sperm parameters, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling test to evaluate DNA fragmentation and fluorescence in situ hybridization or immunohistochemistry for HPV were performed on semen samples from infected patients (n= 22), control subjects (n= 13) and on pooled control sperm samples incubated with HPV16-L1 (HPV capsid), before and after direct swim-up and modified swim-up (with added Heparinase-III). Moreover, cytofluorimetry for HPV detection was performed in pooled sperm pre- and post-incubation with HPV 16-L1 before and after direct and modified swim-up. Statistical analysis was performed with a two-tailed Students t-test. RESULTS Direct swim-up reduces the number of HPV-infected sperm by ~24% (P< 0.01), while modified swim-up is able to remove completely HPV DNA both from naturally and artificially infected sperm. Enzymatic treatment with Heparinase-III tended to decrease sperm motility, viability and DNA integrity but the effects were not significant. CONCLUSIONS This study shows that Heparinase-III treatment seems not to affect spermatozoa in vitro and suggests that this treatment should be investigated further as a means of preparing sperm from patients who are infected with HPV in order to reduce the risk of HPV infection when using assisted reproduction techniques.

Association, prevalence, and clearance of human papillomavirus and antisperm antibodies in infected semen samples from infertile patients.

OBJECTIVE To evaluate prevalence, association, and clearance of human papillomavirus (HPV) and antisperm antibodies (ASAs) in infected semen samples from infertile patients. DESIGN Cross-sectional clinical study. SETTING Andrology and microbiology sections at a university hospital. PATIENT(S) Three groups of subjects: 61 infertile patients with HPV semen infection, 104 noninfected infertile subjects, and 92 control subjects. INTERVENTION(S) Semen analysis, spermMar test, fluorescence in situ hybridization for sperm aneuploidy and for HPV, and immunofluorescence for HPV 16-L1 and immunoglobulins (IgA, IgG, and IgM) determination. MAIN OUTCOME MEASURE(S) Association of sperm procedures, HPV sperm infection, sperm aneuploidies, and sperm ASAs. RESULT(S) Infertile patients with HPV semen infection showed high percentages of ASAs. In these patients HPV sperm infection was associated with lower sperm motility, which was worse in subjects with ASAs. No alterations of sperm chromosomes were observed. To obtain a significant clearance of both HPV sperm infection and ASAs at least 24 months of follow-up were needed. CONCLUSION(S) Human papillomavirus has been recently suggested to have an important role in male infertility. This study demonstrated that HPV sperm infection can be long lasting and frequently associated with ASAs that may further reduce male fertility. Infertile patients with positive spermMar test results should be considered for investigation for HPV, especially if they are candidates for assisted reproduction.

Human papillomavirus proteins are found in peripheral blood and semen Cd20+ and Cd56+ cells during Hpv-16 semen infection

BackgroundHuman papillomavirus (HPV) currently represents an important risk factor for cancer development and infertility in humans. Whilst binding of HPV to spermatozoa has been associated with male infertility, an investigation about the presence of HPV-DNA in non-spermatozoal semen cells is lacking. Previous findings documented the presence of HPV in peripheral blood leukocytes. The aim of this study was to investigate the expression of HPV markers in semen and blood leukocytes during HPV-16 infection.MethodsA total of 32 subjects, 16 patients affected by HPV-16 semen infection and 16 controls, were evaluated in our andrological centre and enrolled in the study. Semen non-spermatozoal cells from all subjects were isolated and evaluated for the expression of HPV-16 markers (DNA and L1, E6 proteins) and further characterized for their molecular phenotype. Analogue determination was performed on peripheral blood mononuclear cells.ResultsThe presence of HPV-DNA by FISH analysis in a round cell population from semen, confirmed to be CD45+ leukocytes, was observed. These HPV-DNA containing-cells also displayed HPV-16-E6 and HPV-16-L1 viral proteins and, upon further investigation, were found to be CD20+ and CD56+, likely phenotypes of B cells and natural killer cells (NK) respectively. In 25% of the patient group, a very small population of peripheral blood mononuclear cells was found to be positive for HPV-DNA via FISH. These cells displayed the CD20+ and CD56+ phenotype alike. None of the control subjects displayed HPV-DNA in either semen or peripheral blood.ConclusionConsidering the role of CD20+ and CD56+ cell populations in the antiviral immune response, the detection of HPV markers on leukocytes may reflect the presence of virus particles within the endosomal compartment. However, the presence of HPV markers in circulating mononuclear cells raise concerns about the risk of developing cancers to distal organs.

Increased Levels of Osteocalcin-Positive Endothelial Progenitor Cells in Patients Affected by Erectile Dysfunction and Cavernous Atherosclerosis

INTRODUCTION Erectile dysfunction (ED) was shown to be the expression of a systemic vascular disease that can precede coronary artery disease of some years. Endothelial progenitor cells (EPCs) are a population of circulating cells with endothelial-regenerative potential that may be reduced in ED and coronary patients. Recently, increased levels of osteocalcin (OCN)-positive EPC have been reported in coronary patients. AIM Investigate the correlation between OCN-positive EPC and cavernous atherosclerotic lesion in ED patients. METHODS A total of 35 subjects (20 ED patients and 15 controls) were evaluated in our andrological center and enrolled in the study. MAIN OUTCOME MEASURE All subjects underwent routine clinical examination. Patients were also evaluated with high resolution echo color doppler of penile districts (intima media thickness [IMT] before and after intracavernous alprostadil injection) and circulating levels of progenitor cells (PC), EPC, and OCN-positive fraction of EPC. RESULTS A progressive reduction of circulating EPC with the severity of cavernous artery atherosclerosis was found. Conversely circulating OCN-positive EPC levels undergo to a significant increase with cavernous atherogenesis progression. CONCLUSIONS OCN-positive EPC levels in association with penile-color Doppler ultrasound evaluation of cavernous IMT could be predictive markers of subsequent coronary artery disease in ED patients.

Reduced Endothelial Progenitor Cell Number and Function in Inflammatory Bowel Disease: A Possible Link to the Pathogenesis

OBJECTIVES:Circulating endothelial progenitor cells (EPCs) are essential for endothelial repair and vascular healing. Patients with inflammatory bowel disease (IBD) may suffer from endothelial dysfunction. Reduced EPC number, impaired mobilization, or increased EPC apoptosis may be crucial in this phenomenon. The aim of our study was to investigate the number and function of EPCs in patients with IBD and to assess their endothelial function.METHODS:In 100 IBD patients (47 ulcerative colitis (UC) and 53 Crohns disease (CD)) and 50 healthy controls, EPC number, CXC motif receptor 4 (CXCR4) expression, the percentage of apoptotic circulating EPCs, and the number of colony-forming units were evaluated. Endothelial dysfunction was assessed by luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone levels, and in a subgroup of patients, brachial artery flow-mediated dilation (FMD) was measured. Kruskal–Wallis ANOVA (analysis of variance), Mann–Whitney U two-tailed, and Spearmans rank correlation tests were used to assess differences.RESULTS:EPC number was significantly lower in UC patients (39.6 (95% confidence interval (95% CI): 30.7–48.6)) and in CD patients (43.1 (95% CI: 35.9–50.4)) than in healthy controls (97.1 (95% CI: 88.3–105.9)), (P<0.001). LH and FSH levels and CXCR4 expression on EPCs did not significantly differ from controls. Testosterone concentrations and FMD were lower in UC patients. Number of apoptotic EPCs was higher in both UC and CD patients with an impaired ability to generate colony in vitro.CONCLUSIONS:We hypothesize that in IBD patients, apoptosis contributes to the reduction of circulating EPC number and to their ability to proliferate in vitro. As this condition represents a risk factor for cardiovascular disease, endothelial function should be evaluated in these patients.