Himanshu Arora

Subcutaneous Leydig Stem Cell Autograft: A Promising Strategy to Increase Serum Testosterone

Exogenous testosterone therapy can be used to treat testosterone deficiency; however, it has several adverse effects including infertility due to negative feedback on the hypothalamic–pituitary–gonadal (HPG) axis. Leydig stem cell (LSC) transplantation could provide a new strategy for treating testosterone deficiency, but clinical translatability of injecting stem cells inside the testis is not feasible. Here, we explore the feasibility of subcutaneously autografting LSCs in combination with Sertoli and myoid cells to increase testosterone. We also studied whether the grafted LSCs can be regulated by the HPG axis and the molecular mechanism behind this regulation. LSCs were isolated from the testes of 12‐week‐old C57BL/6 mice, and subcutaneously autografted in combination with Sertoli cells and myoid cells. We found that LSCs alone were incapable of self‐renewal and differentiation. However, in combination with Sertoli cells and myoid cells, LSCs underwent self‐renewal as well as differentiation into mature Leydig cells. As a result, the recipient mice that received the LSC autograft showed testosterone production with preserved luteinizing hormone. We found that testosterone production from the autograft was regulated by hedgehog (HH) signaling. Gain of function and loss of function study confirmed that Desert HH (DHH) agonist increased and DHH antagonist decreased testosterone production from autograft. This study is the first to demonstrate that LSCs, when autografted subcutaneously in combination with Sertoli cells and myoid cells, can increase testosterone production. Therefore, LSC autograft may provide a new treatment for testosterone deficiency while simultaneously preserving the HPG axis. Stem Cells Translational Medicine 2019;8:58–65

Alterations of tumor microenvironment by nitric oxide impedes castration-resistant prostate cancer growth

Significance This study presents insights into the underexplored areas of castration-resistant prostate cancer (CRPC) therapeutics—the role of nitric oxide (NO) in CRPC reduction through its microenvironment. Results of this study provide important information on the tumor reduction capabilities of increased NO levels and its mechanistic aspect and demonstrates the potential long-term efficacy of NO on CRPC. An in-depth understanding of how NO affects the tumor microenvironment will allow development of chemotherapeutics based on NO for a CRPC cure. Immune targeted therapy of nitric oxide (NO) synthases are being considered as a potential frontline therapeutic to treat patients diagnosed with locally advanced and metastatic prostate cancer. However, the role of NO in castration-resistant prostate cancer (CRPC) is controversial because NO can increase in nitrosative stress while simultaneously possessing antiinflammatory properties. Accordingly, we tested the hypothesis that increased NO will lead to tumor suppression of CRPC through tumor microenvironment. S-nitrosoglutathione (GSNO), an NO donor, decreased the tumor burden in murine model of CRPC by targeting tumors in a cell nonautonomous manner. GSNO inhibited both the abundance of antiinflammatory (M2) macrophages and expression of pERK, indicating that tumor-associated macrophages activity is influenced by NO. Additionally, GSNO decreased IL-34, indicating suppression of tumor-associated macrophage differentiation. Cytokine profiling of CRPC tumor grafts exposed to GSNO revealed a significant decrease in expression of G-CSF and M-CSF compared with grafts not exposed to GSNO. We verified the durability of NO on CRPC tumor suppression by using secondary xenograft murine models. This study validates the significance of NO on inhibition of CRPC tumors through tumor microenvironment (TME). These findings may facilitate the development of previously unidentified NO-based therapy for CRPC.

A rare ANOS1 variant in siblings with Kallmann syndrome identified by whole exome sequencing

Kallmann syndrome is a rare genetic condition causing congenital hypogonadotropic hypogonadism. It presents with delayed puberty, anosmia, and infertility. Here, we set out to identify a causative DNA variant for Kallmann syndrome in two affected brothers of Hispanic ancestry. The male siblings presented with a clinical diagnosis of Kallmann syndrome (anosmia, delayed puberty, azoospermia, and undetectable luteinizing hormone and follicle stimulating hormone levels). Genetic variations were investigated by whole exome sequencing. Potentially pathogenic variants were filtered and prioritized followed by validation by Sanger sequencing in the two brothers and their mother. A pathogenic variant was identified in the ANOS1 gene on the X chromosome: c.1267C>T; both brothers were hemizygous, and their mother was heterozygous for the variant. The variant is a single nucleotide change that introduces a stop codon in exon 9 (p.R423*), likely producing a truncated variant of the protein. This variant has only been reported twice in the literature, in the setting of finding genetic causes for other conditions. This result supports the clinical value of whole exome sequencing for identification of genetic pathogenic variants. Genetic diagnosis is the essential first step for genetic counseling, preimplantation diagnosis, and research for a potential treatment.

PD08-08 S-NITROSOGLUTATHIONE REDUCTASE (GSNOR) KNOCKOUT MICE: A NOVEL MODEL OF MALE INFERTILITY

model, Lecithin: Retinol Acyltransferase (LRAT) knockout (KO) mouse was used to answer our question. METHODS: 9 LRAT KO animals were fed either Vitamin A sufficient (ASuf) or Vitamin A deficient (ADef) diets for 8wks, with the latter known to produce SCO. H&E of testicular slides was used to assess histology. Immunofluorescence (IF) with antibodies against SYCP3 (marker of spermatocytes) was used to confirm loss of meiotic cells in LRAT KO Adef mice. RNAseq and smallRNA sequencing was performed using total RNA extracted from testes. Sequencing results were processed using JMP Genomics. Expression levels of GFRa1, PLZF, SCYP3, PRM1, TNP, CLU, and VIM were used to evaluate loss of different germ cell populations in the Adef group, and to normalize the data to the number of Sertoli cells. MicroRNA202-5p expression (Sertoli specific), miR-34c (germ cell specific) and let-7 (ubiquitous) were measured from sequencing data and further confirmed using QRT-PCR. Results were statistically significant at FRD1⁄40.01 RESULTS: 3 mice were evaluated in each respective group, LRAT Asuf and ADef conditions, at both 6 and 8wk time points. Histology, IF, and sequencing data demonstrated that spermatogenesis was present in all groups except for LRAT ADef mice at 8 weeks. Histology revealed heterogeneity, with most tubules resembling SCO in LRAT ADef testes. Normal spermatogenesis was observed in LRAT KO Asuf mice, and hypo spermatogenesis was observed in LRAT KO ADef mice at 6wks. There was no significant change in expression levels of miR202-5p between LRAT ASuf an ADef groups at 8wks. However, expression of miR34c was significantly decreased in the LRAT Adef group. Let-7 expression remained same. CONCLUSIONS: Loss of meiotic germ cells in LRAT KO ADef mice did not result in loss of miRNA-202-5p expression when compared to controls despite development of predominantly SCO histology. Our results provide strong evidence that the observed loss of expression of miRNA202-5p in men with SCO is not due to the primary loss of germ cells but is a result of primary miRNA-202-5p dysfunction in Sertoli cells.

MP81-04 LEYDIG STEM CELL ISOLATION AND DIFFERENTIATION FROM HUMAN TESTIS BIOPSIES: POTENTIAL MODALITY TO INCREASE SERUM TESTOSTERONE

INTRODUCTION AND OBJECTIVES: This study aimed to examine whether there is an independent association between obstructive sleep apnea (OSA) and other sleep indices using polysomnography data and erectile dysfunction (ED) in a representative cohort of middle-aged to elderly men. METHODS: Data were drawn from a randomly-selected, community-dwelling cohort of men aged 1⁄435y at recruitment (2002e5). Of these, n1⁄4734 men with no prior OSA diagnosis who underwent full inhome polysomnography (Embletta X100; 2010-11) and had complete ED measures (Global Impotence Rating) were selected for the analytic sample (mean age (SD): 60.8 (10.9)). Uniand multi-adjusted regression models of ED were fitted against PSG measures, along with related covariates. RESULTS: Of the men examined, 24.7% (n1⁄4181) had ED, most notably in men aged over 65 years (p<0.001). Given an observed age interaction (p1⁄40.005), analyses were repeated in age-stratified samples (<65; 65+ years). In men <65 years, only severe OSA was found to have an association with ED (2.01; 1.13-4.69) in unadjusted models, however this effect was attenuated after adjustment. For men 65+ years, an independent association with ED was found for AHI (1.55;1.02-2.36), moderate (1.79;1.18-2.43) and severe (4.84;2.569.93) OSA, and ODI (both continuous (1.48;1.03-1.99) and >16 secs (2.79;1.23-6.32)). The effect of AHI on ED was shown to be primarily mediated through ODI (63.4%, Sobel p value1⁄40.29). CONCLUSIONS: In younger, community-based men there appeared no independent relationship between objective measures of sleep and ED. However, there appears a strong, independent relationship between OSA, ODI and ED in men aged 65 and over.

PD08-11 LEYDIG STEM CELL AUTOGRAFT IN MICE: A NOVEL APPROACH TO INCREASE SERUM TESTOSTERONE WHILE PRESERVING FERTILITY

INTRODUCTION AND OBJECTIVES: Sensitivity of application of molecular transcripts as non-invasive biomarkers for assessment of spermatogenesis in cases with non-obstructive azoospermia is hindered; mostly by the restricted number of gametocytes in seminal fluid. In addition, histopathological examination of testicular biopsies of those cases is sometimes impeded by the focal distribution of spermatogenesis. The aim of the present work was to investigate the potential application of DDX4 gene expression in cell free seminal mRNA as a non-invasive biomarker for the identification of the presence of germ cells in men with non-obstructive azoospermia and to correlate this factor with other predictors of spermatogenesis in non-obstructive azoospermia, namely: testicular biopsy. METHODS: Thirty-nine infertile males diagnosed as nonobstructive azoospermia (NOA) by means of testicular biopsy, were enrolled in this study. A group of twenty-eight normospermic fertile males served as a control group. The study was approved by the Ethics Committee. All patients provided informed consent to participate in this study. Thorough history taking and physical examination were carried out. Semen was collected by masturbation after 3-5 days of abstinence and was analyzed according to World Health Organization guidelines, 2010. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone level were all assayed on AdviaTM Centaur Immunoassay System. Two male reproductive organs-specific genes were chosen; DDX4 which is a germ cell-specific gene,and TGM4 (transglutaminase 4) which is a prostate specific gene which was included as a control gene. Gene expression for both DDX4 and TGM4 genes were performed using TaqMan Gene Expression Assays. Gene expression was assessed using the 2-??Ct method. RESULTS: Thirty-nine azoospermic males participated in the present study. A group of 28 normospermic fertile males served as a control group. Both groups were matched for age. Level of both serum FSH and LH was statistically higher, while serum testosterone showed statistically lower values in azoospermic males (p1⁄40.001). Histopathological examination of testicular biopsies categorized azoospermic males into 20.5% (n1⁄4 8) with maturation arrest (MA), 17.9 % (n1⁄4 7) with incomplete Sertoli Cell Only Syndrome (icSCOS) and 61.5 % (n1⁄424) with complete Sertoli Cell Only Syndrome (cSCOS).In patients with azoospermia, TGM4 gene was detected in 100% of semen samples. Positivity for DDX4 gene was detected in 17 out of 39 males with NOA which was attributed to MA in 35.3% (n1⁄46/17), icSCOS in 23.5% (n1⁄44/ 17) and cSCOS in 41.2% (n1⁄47/17). Both DDX4 and TGM4 genes were consistently detected in semen samples of all control subjects. CONCLUSIONS: Detection of germ cell non stage specific gene mRNA in cell free seminal plasma is a non-invasive screening tool of non-obstructive azoospermia. It is a good positive diagnostic tool but not a good negative one, as some DDX4 negative cases were proved by testicular biopsy to be cases of maturation arrest and not cases of complete Sertoli Cell Only Syndrome. Moreover, it could detect foci of spermatogenesis in casesb histopathologically diagnosed as complete Sertoli Cell Only Syndrome.