Extragonadal function of follicle-stimulating hormone: Evidence for a role in endothelial physiology and dysfunction.

AIMS: Follicle-stimulating hormone (FSH) plays a fundamental role in reproduction stimulating ovarian folliculogenesis, Sertoli cells function and spermatogenesis. However, the recent identification of FSH receptor (FSHR) also in extra-gonadal tissues has suggested that FSH activity may not be limited only to fertility regulation, with conflicting results on the possible role of FSH in endothelial cells. The aim of this study was to investigate FSH role on endothelial function in Human Umbilical Vein Endothelial Cells (HUVECs). RESULTS: Endothelial Nitric oxide synthase (eNOS) expression, eNOS phosphorylation and Nitric Oxide (NO) production resulted increased after the stimulation of HUVEC with recombinant human FSH (rhFSH) at 3.6x103 ng/ml, with increasing Calcium release from intracellular stores. Furthermore, IP3 production increased after rhFSH stimulation despite PTX treatment and NFAT1 was observed prevalently in nucleus. We observed a statistical difference between untreated cells and cells stimulated with 0.36x103 ng/ml and between cells stimulated with 0.36x103 ng/ml and cells stimulated with 1.8x103 ng/ml at 4 and 8 h by Wound healing assay, respectively. Furthermore, a higher cellular permeability was observed in stimulated cells, with atypical VE-cadherin distribution, as well as filamentous actin. CONCLUSIONS: Our findings suggest that FSH at high concentrations elicits a signalling that could compromise the endothelial membrane. Indeed, VE-cadherin anomalies may severely affect the endothelial barrier, resulting in an increased membrane permeability. Although NO is an important vasodilatation factor, probably an excessive production could impact on endothelial functionality, partially explaining the increased risk of cardiovascular diseases in menopausal women and men with hypogonadism.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Study of novel androgen receptor V770 variant in androgen insensitivity syndrome patients reveals the transitional state of the androgen receptor ligand binding domain homodimer.

We report the discovery of the androgen receptor missense mutation V770D, that was found in two sisters suffering from complete androgen insensitivity. Experimental validation of AR V770 variants demonstrated that AR V770D was transcriptionally inactive due to the inability to dimerize and a reduced ligand binding affinity. The more conservative AR V770A variant showed a dimerization defect at low levels of DHT with a partial recovery of the transcriptional activity and of the receptor's ability to dimerize when increasing the DHT levels. With V770 located outside of the proposed LBD dimerization interface of the AR LBD homodimer crystal structure, the effects of the V770A mutation on AR dimerization were unexpected. We therefore explored whether the AR LBD dimerization interface would be better described by an alternative dimerization mode based on available human homodimeric LBD crystal structures of other nuclear receptors. Superposition of the monomeric AR LBD in the homodimeric crystal structures of GR, PR, ER, CAR, TRß, and HNF-4α showed that the GR-like LBD dimer model was energetically the most stable. Moreover, V770 was a key energy residue in the GR-like LBD dimer while it was not involved in the stabilization of the AR LBD homodimer according to the crystal structure. Additionally, the observation that 4 AIS mutations impacted the stability of the AR LBD dimer while 16 mutations affected the GR-like LBD dimer, suggested that the AR LBD dimer crystal is a snapshot of a breathing AR LBD homodimer that can transition into the GR-like LBD dimer model.

Mutational Screening of Androgen Receptor Gene in 8224 Men of Infertile Couples.

CONTEXT: Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity. OBJECTIVE: The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males. METHODS: A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses. RESULTS: We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic. CONCLUSION: This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

Comparative Evaluation of the Effects of Legacy and New Generation Perfluoralkyl Substances (PFAS) on Thyroid Cells In Vitro.

Background: Per- and poly-fluorinated alkyl substances (PFAS) are environment-persitent emerging endocrine disrupting chemicals raising health concerns worldwide. Exposure to PFAS has been associated with the imbalance of thyroid hormones. However, available studies addressing the cell mechanism underlying thyroid disrupting feature of legacy PFAS, such as perfluoro-octanoic acid (PFOA), perfluoro-octane-sulfonic acid (PFOS), and the new generation substitutes, such as C6O4, are still lacking. In this study the potential disrupting effect of PFOA, PFOS, and C6O4 on a murine thyroid cell model was assessed. Methods: A rat FRTL-5 cell line was used as the normal thyroid follicular cell model. Cell iodide-uptake, induced by thyroid stimulating hormone (TSH), was used to assess the functional impact of PFAS exposure on cell function. Tetrazolium salt-based cell viability assay and merocyanine 540-based cell staining were used to address the possible involvement of cell toxicity and membrane biophysical properties on altered cell function. The possible direct interaction of PFAS with TSH-receptor (TSH-R) was investigated by computer-based molecular docking and analysis of molecular dynamics. Evaluation of intracellular cAMP levels and gene expression analysis were used to validate the direct impairment of TSH-R-mediated downstream events upon PFAS exposure. Results: Different from PFOS or C6O4, exposure to PFOA at a concentration ≥ 10 ng/mL was associated with significant impairment of the iodide uptake upon TSH stimulation (respectively: basal 100.0 ± 19.0%, CTRL + TSH 188.9 ± 7.8%, PFOA 10 ng/mL + TSH 120.4 ± 20.9%, p= 0.030 vs CTRL + TSH; PFOA 100 ng/mL + TSH 115,6 ± 12,3% p= 0.017 vs CTRL + TSH). No impairment of cell viability or membrane stability was observed. Computational analysis showed a possible direct differential interaction of C6O4, PFOA, and PFOS on a same binding site of the extracellular domain of TSH-R. Finally, exposure to PFOA was associated with a significant reduction of downstream intracellular cAMP levels and both sodium-iodide transporter and thyroperoxidase gene expression upon TSH-R stimulation. Conclusions: Our data suggest that legacy and new generation PFAS can differentially influence TSH dependent signaling pathways through the direct interaction with TSH-R.

Cutaneous Melanoma and Hormones: Focus on Sex Differences and the Testis.

Cutaneous melanoma, the most aggressive type of skin cancer, remains one the most represented forms of cancer in the United States and European countries, representing, in Australia, the primary cause of cancer-related deaths. Recently, many studies have shown that sex disparities previously observed in most cancers are particularly accentuated in melanoma, where male sex is consistently associated with an increased risk of disease progression and a higher mortality rate. The causes of these sex differences rely on biological mechanisms related to sex hormones, immune homeostasis and oxidative processes. The development of newer therapies, such as immune checkpoint inhibitors (ICIs) (i.e., anti-PD-1 and anti-CTLA-4 monoclonal antibodies) has dramatically changed the treatment landscape of metastatic melanoma patients, though ICIs can interfere with the immune response and lead to inflammatory immune-related adverse events (irAEs). Recently, some studies have shown a potential adverse influence of this immunotherapy treatment also on male fertility and testicular function. However, while many anticancer drugs are known to cause defects in spermatogenesis, the effects of ICIs therapy remain largely unknown. Notwithstanding the scarce and conflicting information available on this topic, the American Society of Clinical Oncology guidelines recommend sperm cryopreservation in males undergoing ICIs. As investigations regarding the long-term outcomes of anticancer immunotherapy on the male reproductive system are still in their infancy, this review aims to support and spur future research in order to understand a potential gonadotoxic effect of ICIs on testicular function, spermatogenesis and male fertility.

Impairment of human dopaminergic neurons at different developmental stages by perfluoro-octanoic acid (PFOA) and differential human brain areas accumulation of perfluoroalkyl chemicals.

Perfluoroalkyl substances (PFASs) are synthetic chemicals widely used in industrial and consumer products. The environmental spreading of PFASs raises concerns for their impact on human health. In particular, the bioaccumulation in humans due to environmental exposure has been reported also in total brain samples and PFAS exposure has been associated with neurodevelopmental disorders. In this study we aimed to investigate the specific PFAS bioaccumulation in different brain areas. Our data reported major accumulation in the brainstem region, which is richly populated by dopaminergic neurons (DNs), in brain autopsy samples from people resident in a PFAS-polluted area of Italy. Since DNs are the main source of dopamine (DA) in the mammalian central nervous system (CNS), we evaluated the possible functional consequences of perfluoro-octanoic acid (PFOA) exposure in a human model of DNs obtained by differentiation of human induced pluripotent stem cells (hiPSCs). Particularly, we analyzed the specific effect of the exposure to PFOA for 24 h, at the concentration of 10 ng/ml, at 3 different steps of dopaminergic differentiation: the neuronal commitment phase (DP1), the neuronal precursor phase (DP2) and the mature dopaminergic differentiation phase (DP3). Interestingly, compared to untreated cells, exposure to PFOA was associated with a reduced expression of Tyrosine Hydroxylase (TH) and Neurofilament Heavy (NFH), both markers of dopaminergic maturation at DP2 phase. In addition, cells at DP3 phase exposed to PFOA showed a severe reduction in the expression of the Dopamine Transporter (DAT), functionally involved in pre-synaptic dopamine reuptake. In this proof-of-concept study we show a significant impact of PFOA exposure, mainly on the most sensitive stage of neural dopaminergic differentiation, prompting the way for further investigations more directly relevant to risk assessment of these chemicals.

Identification of Rare LRP5 Variants in a Cohort of Males with Impaired Bone Mass.

Osteoporosis is the most common bone disease characterized by reduced bone mass and increased bone fragility. Genetic contribution is one of the main causes of primary osteoporosis; therefore, both genders are affected by this skeletal disorder. Nonetheless, osteoporosis in men has received little attention, thus being underestimated and undertreated. The aim of this study was to identify novel genetic variants in a cohort of 128 males with idiopathic low bone mass using a next-generation sequencing (NGS) panel including genes whose mutations could result in reduced bone mineral density (BMD). Genetic analysis detected in eleven patients ten rare heterozygous variants within the LRP5 gene, which were categorized as VUS (variant of uncertain significance), likely pathogenic and benign variants according to American College of Medical Genetics and Genomics (ACMG) guidelines. Protein structural and Bayesian analysis performed on identified LRP5 variants pointed out p.R1036Q and p.R1135C as pathogenic, therefore suggesting the likely association of these two variants with the low bone mass phenotype. In conclusion, this study expands our understanding on the importance of a functional LRP5 protein in bone formation and highlights the necessity to sequence this gene in subjects with idiopathic low BMD.

Exposure to Perfluoro-Octanoic Acid Associated With Upstream Uncoupling of the Insulin Signaling in Human Hepatocyte Cell Line.

Perfluoro-alkyl substances (PFAS) are chemical pollutants with prevalent stability and environmental persistence. Exposure to PFAS, particularly perfluoro-octanoic acid (PFOA), has been associated with increased diabetes-related cardiovascular mortality in subjects residing areas of high environmental contamination, however the exact pathogenic mechanism remains elusive. Here we used HepG2 cells, an in vitro model of human hepatocyte, to investigate the possible role of PFOA exposure in the alteration of hepatic glucose metabolism. HepG2 cells were exposed for 24 hours to PFOA at increasing concentration from 0 to 1000 ng/mL and then stimulated with 100 nm Insulin (Ins). The consequent effect on glycogen synthesis, glucose uptake and Glut-4 glucose transporter translocation was then evaluated by, respectively, Periodic Acid Schiff (PAS) staining, 2-deoxyglucose (2-DG) uptake assay and immunofluorescence. Exposure to PFOA was associated with reduced glycogen synthesis and glucose uptake, at concentration equal or greater than, respectively, 0,1 ng/mL and 10 ng/mL, with parallel impaired membrane translocation of Glut-4 upon Ins stimulation. Western blot analysis showed early uncoupling of Insulin Receptor (InsR) activation from the downstream Akt and GSK3 phosphorylation. Computational docking analysis disclosed the possible stabilizing effect of PFOA on the complex between InsR and GM3 ganglioside, previously shown to be associated with the low grade chronic inflammation-related insulin resistance. Consistently, long term treatment with glucosyl-ceramide synthase inhibitor PDMP was able to largely restore glycogen synthesis, glucose uptake and Glut-4 translocation upon Ins stimulation in HepG2 exposed to PFOA. Our data support a novel pathogenic mechanism linking exposure to PFOA to derangement of hepatocyte cell metabolism.

TERRA: A Novel Biomarker of Embryo Quality and Art Outcome.

Telomeres are considered to be an internal biological clock, and their progressive shortening has been associated with the risk of age-related diseases and reproductive alterations. Over recent years, an increasing number of studies have focused on the association between telomere length and fertility, identifying sperm telomere length (STL) as a novel biomarker of male fertility. Although typically considered to be repeated DNA sequences, telomeres have recently been shown to also include a long non-coding RNA (lncRNA) known as TERRA (telomeric repeat-containing RNAs). Interestingly, males with idiopathic infertility show reduced testicular TERRA expression, suggesting a link between TERRA and male fertility. The aim of this study was to investigate the role of seminal TERRA expression in embryo quality. To this end, STL and TERRA expression were quantified by Real Time qPCR in the semen of 35 men who underwent assisted reproductive technologies (ART) and 30 fertile men. We found that TERRA expression in semen and STL was reduced in patients that underwent ART (both p < 0.001). Interestingly, TERRA and STL expressions were positively correlated (p = 0.010), and TERRA expression was positively associated with embryo quality (p < 0.001). These preliminary findings suggest a role for TERRA in the maintenance of sperm telomere integrity during gametogenesis, and for the first time, TERRA expression was found as a predictive factor for embryo quality in the setting of assisted reproduction.

E2F1 copy number variations in germline and breast cancer: a retrospective study of 222 Italian women.

BACKGROUND: Breast cancer is the most common neoplasia among women in developed countries. The risk factors of breast cancer can be distinguished in modifiable and unmodifiable factors and, among the latter, genetic factors play a key role. Copy number variations (CNVs) are genetic variants that are classified as rare when present in less than 1% of the healthy population. Since rare CNVs are often cause of diseases, over the last years, their contribution in carcinogenesis has become a relevant matter of study. E2F1 is a transcriptional factor that plays an important role in regulating cell cycle and apoptosis. Its double and conflicting role is the reason why it acts both as oncogene and as tumour suppressor, depending on cell context. Since anomalies in expression or in number of copies of E2F1 have been related to several cancers, we aimed to study number of germline copies of E2F1 in women with breast cancer in order to better elucidate their contribution as predisposing factor to this tumour. METHODS: We performed, hence, a retrospective study on 222 Italian women with breast cancer recruited from October 2002 to December 2007. TaqMan CNV assay and Real-Time PCR were carried out to analyse, respectively, E2F1 CNV and E2F1 expression in the subjects of the study. Chi square test or Fisher's exact test and Student's t-test were used to calculate the frequency of CNVs and differences in continuous variables between groups, respectively. RESULTS: Intriguingly, we found that 10/222 (4.5%) women with breast cancer had more copies than controls (0/200, 0%), furthermore, the number of copies positively correlated with E2F1 gene expression in breast cancer tissue, suggesting that the constitutive gain of the gene could translate into an increased risk of genomic instability. Additionally, we found that altered E2F1 copies were present prevalently in the patients with contralateral breast cancer (20%) and all of them had a positive family history, both typically associated with hereditary cancer. CONCLUSIONS: Our findings suggest that copy number variations of E2F1 might be a susceptibility factor for breast cancer, however, further studies on large cohorts are to be performed in order to better delineate the phenotype linked to the gain of E2F1 copies.

Caution in the management of SARS-CoV-2 infection in males.

The coronavirus 2 (SARS-CoV-2) pandemic carries clinical, economic, and social burdens that are currently being disclosed. The key steps of virus life cycle have been recently clarified, highlighting the role of host type 2 angiotensin-converting enzyme (ACE2) and TMPRSS2 serine protease in virus-cell binding and entry, respectively. Importantly, major concerns derive from the androgen-dependent tissue-expression of both TMPRSS2 and ACE2, suggesting a differential clinical course of the infection between genders. In agreement with this model, available epidemiological data show that the disease in males has an higher risk to display an heavier pattern and associates with both an increased access to critical care unit and higher mortality rate. In this opinion article, available evidence linking the androgen activity with the gender differences observed in SARS-CoV-2 infection are discussed, hypothesizing possible therapeutic approaches in male based on the disruption of androgen signaling. On these bases, gender-specific recommendations for the management of male patients affected by SARS-CoV-2 infection are warmly suggested, in order to improve the clinical course of the disease.

A novel genetic variant in DNAI2 detected by custom gene panel in a newborn with Primary Ciliary Dyskinesia: case report.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a highly heterogeneous genetic disorder caused by defects in motile cilia. The hallmark features of PCD are the chronic infections of the respiratory tract, moreover, clinical manifestations include also laterality defects and risk of male infertility. Clinical phenotypes of PCD are the result of mutations in genes encoding components of axonema or factors involved in axonemal assembly. Recent studies have identified over 45 PCD-associated genes, therefore, molecular analysis represents a powerful diagnostic tool to confirm and uncover new genetic causes of this rare disease. CASE PRESENTATION: Here, we describe a female infant of Moroccan origin with normal pressure hydrocephalus (NPH) in addition to most common PCD symptoms. Transmission Electron Microscopy (TEM) and molecular tests, such as a Next generation Sequencing panel and a custom array CGH, were performed for diagnosis of PCD. TEM revealed outer dynein arm (ODA) defects, whilst molecular analyses detected a novel 6,9 kb microdeletion in DNAI2 gene. CONCLUSIONS: Since DNAI2 mutations are very rare, this case report contributes to better delineate the important role of DNAI2 as causative of PCD phenotype, suggesting, furthermore, that the variations in DNAI2 may be as a new genetic risk factor for NPH. Indeed, although the association of hydrocephalus with PCD has been well documented, however, only a small number of human patients show this defect. Furthermore, this study highlights the importance of high-throughput technologies in advancing our understanding of heterogeneous genetic disorders.

Endocrine disruption of vitamin D activity by perfluoro-octanoic acid (PFOA).

Perfluoroalkyl substances (PFAS) are a class of compounds used in industry and consumer products. Perfluorooctanoic acid (PFOA) is the predominant form in human samples and has been shown to induce severe health consequences, such as neonatal mortality, neurotoxicity, and immunotoxicity. Toxicological studies indicate that PFAS accumulate in bone tissues and cause altered bone development. Epidemiological studies have reported an inverse relationship between PFAS and bone health, however the associated mechanisms are still unexplored. Here, we present computational, in silico and in vitro evidence supporting the interference of PFOA on vitamin D (VD). First, PFOA competes with calcitriol on the same binding site of the VD receptor, leading to an alteration of the structural flexibility and a 10% reduction by surface plasmon resonance analysis. Second, this interference leads to an altered response of VD-responsive genes in two cellular targets of this hormone, osteoblasts and epithelial cells of the colorectal tract. Third, mineralization in human osteoblasts is reduced upon coincubation of PFOA with VD. Finally, in a small cohort of young healthy men, PTH levels were higher in the exposed group, but VD levels were comparable. Altogether these results provide the first evidence of endocrine disruption by PFOA on VD pathway by competition on its receptor and subsequent inhibition of VD-responsive genes in target cells.

Development of a novel next-generation sequencing panel for diagnosis of quantitative spermatogenic impairment.

PURPOSE: To develop and assess a novel custom next-generation sequencing (NGS) panel for male infertility genetic diagnosis. METHODS: A total of 241 subjects with diagnosis of idiopathic infertility ranging from azoospermia to normozoospermia were sequenced by a custom NGS panel including AR, FSHB, FSHR, KLHL10, NR5A1, NANOS1, SEPT12, SYCP3, TEX11 genes. Variants with minor allele frequency < 1% were confirmed by Sanger sequencing. RESULTS: Nineteen missense variants were detected in 23 subjects with abnormal sperm count, whilst no variants were identified in normozoospermic men. Of identified variants, we prioritized variants classified as pathogenic and of uncertain significance (VUS) (63.1%, 12/19). No missense variants were found in males with normal seminal parameters (0/67). Therefore, the prevalence of variants was significantly higher in patients with spermatogenic impairment (16/174 vs 0/67, p = 0.007). CONCLUSION: This study confirms the utility to apply NGS panel for infertility diagnosis in order to find new genetic variants potentially linked to male infertility with much higher accuracy than standard tests suggested by guidelines. Indeed, based on biological significance, prevalence in the general population and clinical data of patients, it is plausible that identified variants in this study might be linked to quantitative spermatogenic impairment, although further studies are needed.

Testosterone is sequestered in dysfunctional adipose tissue, modifying androgen-responsive genes.

BACKGROUND/OBJECTIVE: The recognized association between male hypogonadism and obesity has multifactorial implications on adipose tissue (AT) physiology. The fat solubility of testosterone (T) suggests a sequestration process in fat depots, leading to reduced circulating levels of T in obesity. Several evidence suggest that steroids play a two-sided inhibitory role on adipogenesis by locally decreasing lipid accumulation and by stimulating lipolysis. The current study investigates T trafficking and activity in dysfunctional AT. SUBJECTS/METHODS: Samples of subcutaneous AT (SAT) were obtained from explants from lipoaspirate plastic surgery in six obese and six normal weight male patients. Experimental procedures on both SAT explants and insulin-resistant (IR) 3T3-L1 adipocytes were performed, including real-time PCR and mass-spectrometry quantification. RESULTS: A significant deregulation of gene responsiveness to androgens in IR cells and obese SAT was observed (all p < 0.05), together with reduced T release after adrenergic stimulation (-10% compared with -55% in lean SAT, p = 0.021). Higher concentrations of intracellular T and estradiol in obese SAT were also observed (2.4 vs. 1.3 ng/g, p = 0.013 and 0.075 vs. 0.22 ng/g, p = 0.004, respectively). Testosterone accumulation resulted in even lower expression in androgen-responsive genes involved in lipolytic and anti-adipogenic pathways from both in vitro and ex vivo experiments. CONCLUSIONS: These results suggest an altered response of dysfunctional fat cells to testosterone stimulation, which normally favors lipolysis and induces an anti-adipogenic effect. The considerable reduction of lipolytic T release after adrenergic stimulation in obese SAT contributes to AT dysfunction, in a feedforward loop further reducing T levels in obese hypogonadal males.

Comparison of NGS panel and Sanger sequencing for genotyping CAG repeats in the AR gene.

BACKGROUND: The androgen receptor (AR) is a nuclear receptor, encoded by the AR gene on the X chromosome. Within the first exon of the AR gene, two short tandem repeats (STR), CAG and GGC, are a source of polymorphism in the population. Therefore, high-throughput methods for screening AR, such as next-generation sequencing (NGS), are sought after; however, data generated by NGS are limited by the availability of bioinformatics tools. Here, we evaluated the accuracy of the bioinformatics tool HipSTR in detecting and quantify CAG repeats within the AR gene. METHOD: The AR gene of 228 infertile men was sequenced using NGSgene panel. Data generated were analyzed with HipSTR to detect CAG repeats. The accuracy was compared with the results obtained with Sanger. RESULTS: We found that HipSTR was more accurate than Sanger in genotyping normal karyotype men (46,XY), however, it was more likely to misidentify homozygote genotypes in men with Klinefelter syndrome (47,XXY). CONCLUSION: Our findings show that the bioinformatics tool HipSTR is 100% accurate in detecting and assessing AR CAG repeats in infertile men (46,XY) as well as in men with low-level mosaicism.

Evaluation of Serum/Urine Genomic and Metabolomic Profiles to Improve the Adherence to Sildenafil Therapy in Patients with Erectile Dysfunction.

Type V-phosphodiesterase-inhibitors (PDE5i) are the first choice drugs in the treatment of erectile dysfunction (ED), being effective in 60-70% of patients. However, approximately 50% of patients per year discontinue the treatment with PDE5i after reporting poor drug efficacy or major adverse drug reactions (ADR). To identify early markers of efficacy/safety for the treatment of ED with PDE5i, the basal clinical characteristics of patients, integrated with metabolomics analysis of serum and urine and genomic data, were here correlated with the PDE5i efficacy and the occurrence of ADR upon administration. Thirty-six males with new diagnosis of ED were consecutively recruited and characterized at baseline for anthropometrics, blood pressure, blood glucose, lipid profile, serum levels of thyroid/sex hormones and erectile function evaluated by IIEF-15 questionnaire. Targeted Next Generation Sequencing (NGS) was applied to genes involved in PDE5i pharmacodynamics and pharmacokinetics. Fasting metabolic profiles of serum and urine were assessed by nuclear magnetic resonance (NMR)-based metabolomics analysis. Patients were prescribed on-demand therapy with Sildenafil oro-dispersible film and followed-up after 3 months from recruitment. Baseline data were compared with IIEF-15 score at follow-up and with the occurrence of ADR recorded by a dedicated questionnaire. Twenty-eight patients were finally included in the analysis. Serum LDL-cholesterol levels were increased in those reporting ADR (143.3 ± 13.2 mg/dl ADR vs. 133.1 ± 12.4 mg/dl No ADR; p = 0.046). NGS data showed that specific variants of PDE11A and CYP2D7 genes were more represented in drug responders (both relative risk = 2.7 [0.9-5.1]; p = 0.04). NMR-based metabolomics showed the highest association between serum LDL-cholesterol metabolites and the occurrence of ADR (Hazard ratio = 17.5; p = 0.019). The association between lipid profile and the ADR pattern suggests major cues in the tailoring of ED therapy with PDE5i.

SHBG141-161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and ß-Langerhans cell lines.

GPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141-161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141-161) domain-peptide (SHBG141-161) on GPRC6A. The binding of SHBG141-161 to GPRC6A and downstream dissociation from Gαi(GDP) protein was computationally modelled. SHBG141-161 was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG141-161 was assessed on Leydig MA-10 and Langerhans ß-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG141-161 was predicted to bind to GPRC6A and to reduce the affinity for Gαi(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141-161 were confirmed by CD and displacement experiments. SHBG141-161 stimulated cell secretion of T and insulin, with dose dependency from 10-13 to 10-11M for T release (respectively P = 0,041 10-13M; P = 0,032 10-12M; P = 0,008 10-11M vs basal) and for 10-12 to 10-10M for insulin (respectively P = 0,041 10-12M; P = 0,007 10-11M; P = 0,047 10-10M; P = 0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG141-161, suggesting agonist specificity. SHBG141-161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.