Fertility Outcomes and Sperm-DNA Parameters in Metastatic Melanoma Survivors Receiving Vemurafenib or Dabrafenib Therapy: Case Report.

Melanoma is a frequent neoplasm in young adult males in reproductive age, 10% of them degenerating into regional and/or distant metastases (MM). The use of BRAF inhibitors (BRAFi) vemurafenib and dabrafenib is effective in MM patients harboring BRAF V600E/K/D mutations. Despite the increased life expectancy in MM patients treated with BRAFi, concerns are raised by the possible side effects and increased risk of gonado- and/or genotoxicity associated with these drugs. However, these aspects are currently under-investigated. Here we report the different fertility outcome in two cases of MM patients, harboring BRAF V600E mutation, that received vemurafenib and dabrafenib respectively. The first patient, 36 years at recruitment in 2015 and seeking fatherhood, had an history of relapsing melanoma since 2002 and undergone to numerous interventions and chemotherapy cycles. In November 2011, following detection of BRAF V600E mutation, a daily treatment with vemurafenib (1,440 mg) was prescribed with preventive gamete cryopreservation. BRAFi was effective in the clinical stabilization of the disease. In 2015, semen evaluation at follow-up showed sperm parameters within the normal range and no signs of alteration of either sperm function or sperm-DNA. On these bases, no contraindications for fatherhood were given. After a month of free intercourses, the 38-year-old partner achieved spontaneous pregnancy with a regular course, normal male fetal karyotype and a full term birth. The second patient, 39 years at recruitment in 2018 and seeking fatherhood, had an history of melanoma since 2012. In 2018, following the evidence of disease relapse and detection of the BRAF V600E mutation, treatment with dabrafenib/trametinib (300 mg/day/2 mg/day) was initiated together with preventive gamete cryopreservation. In 2019, semen evaluation at follow up showed sperm count and motility below the reference values, associated with increased indexes of sperm aneuploidies and sperm DNA fragmentation. Accordingly, access to assisted reproduction technique with cryopreserved spermatozoa was suggested. Differently from dabrafenib that was associated to damage to spermatogenesis, high-dose vemurafenib showed no association with gonadotoxicity and genotoxicity in humans, even at high doses. Although further confirmation are required, our data represent a valued cue in oncofertility counseling to MM patients in addition to preventive cryopreservation.

Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa.

The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31-37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis.

Blood levels, apoptosis, and homing of the endothelial progenitor cells after skin burns and escharectomy.

BACKGROUND: Skin burns are an acute trauma involving an extensive vascular damage and an intense inflammatory response. Bone marrow-derived circulating endothelial progenitor cells (EPC) are known to migrate to sites of neovascularization in response to mediators (vascular endothelial growth factor and stromal cell-derived factor-1) released after trauma and ischemia, to contribute to wound healing, and to increase neovascularization of animal prefabricated flaps. Recent data showed an increase in EPC number in burned patients and a positive correlation between EPC number and total body surface area (TBSA) burnt, but data were limited to the first 5 days after thermal injury. METHODS: By using flow cytometry, we studied EPC (CD34, CD133, CD45, and KDR cells) blood levels, apoptosis, and homing (stromal cell-derived factor-1 receptor expression and CXC chemokine receptor 4) in a 1-month follow-up postburn in 25 patients with ≥15% TBSA burnt, at least grade II burns and escharectomy performed at days 5 to 6, with respect to 31 controls. RESULTS: EPC count at admission showed a positive linear correlation with TBSA burnt. The EPC blood levels of the patients were low (50.7 cells/mL±61.8 cells/mL) immediately after thermal injury, then increased with two peaks, at day 1 (188.3 cells/mL±223.2 cells/mL) and day 12 (253.1 cells/mL±430.7 cells/mL) with respect to controls (95.2 cells/mL±28.5 cells/mL, p<0.05), and then returned to normal levels in 1 month. EPC apoptotic rate and inflammatory parameters paralleled EPC blood count. No significant variations were found in CXC chemokine receptor 4 expression. CONCLUSIONS: Thermal injury and escharectomy seem to induce an intense response in EPC production. In particular, escharectomy could improve physiologic wound repair by increasing EPC levels.

Phosphodiesterase-5 inhibitor tadalafil acts on endothelial progenitor cells by CXCR4 signalling.

It has been proposed that endothelial progenitor cells (EPC), originating from the bone marrow contribute to neo-angiogenesis in vivo by forming endothelial cells. Once released in the bloodstream, EPC home at the site of vascular damage where they participate in endothelium regeneration. In this process CXCR4 plays a key role. Recently we demonstrated that a prolonged therapy with phosphodiesterase-5 (PDE5) inhibitors does improve endothelial function and increases circulating EPC, suggesting a role of PDE5 in EPC physiology. Here we tested the expression of PDE5 and CXCR4 on cultured, circulating, and bone marrow resident EPC, and we studied the effect in vivo and in vitro of PDE5 inhibition after administration of a PDE5 inhibitor (tadalafil) on EPC, in term of CXCR4 expression. We documented that in vivo and in vitro EPC express both PDE5 and CXCR4, and that tadalafil administration induced a significant increase in EPC number and the relative CXCR4 expression. This effect is inhibited by selective CXCR4 antagonist. We thus demonstrated that PDE5 inhibition acts on CXCR4 signalling in EPC and we can suppose an involvement of cGMP second messenger system in both EPC release from the bone marrow and EPC-mediated peripheral re-endothelization.

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.