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Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis.

The co-occurrence of several lymphomas in a patient defines composite/synchronous lymphoma. A common cellular origin has been reported for both contingents of such entities. In the present review, we aimed to gather the available data on composite lymphomas associating a classical Hodgkin lymphoma (cHL) with another lymphoma, to better understand the plasticity of mature B and T-cells. This review highlights that >70% of patients with a composite lymphoma are ≥55 years old, with a male predominance. The most reported associations are cHL with follicular lymphoma or diffuse large B-cell lymphoma, with over 130 cases reported. The cHL contingent is often of mixed cellularity type, with a more frequent focal/weak CD20 expression (30% to 55.6%) compared to de novo cHL, suggesting a particular pathophysiology. Moreover, Hodgkin cells may express specific markers of the associated lymphoma (e.g., BCL2/BCL6 for follicular lymphoma and Cyclin D1 for mantle cell lymphoma), sometimes combined with common BCL2/BCL6 or CCND1 rearrangements, respectively. In addition, both contingents may share similar IgH/IgK rearrangements and identical pathogenic variants, reinforcing the hypothesis of a common clonal origin. Finally, cHL appears to be endowed with a greater plasticity than previously thought, supporting a common clonal origin and a transdifferentiation process during lymphomagenesis of composite lymphomas.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Histopathology is the gold standard for distinguishing between colonization and fungal infection, but it does not provide a precise diagnosis of genera/species. However, if the culture is negative or if no specimen is sent to the Mycology laboratory, only the specimen sent to the Pathology department is available. Formalin fixation and paraffin embedding (FFPE) cause DNA damage, making it difficult to perform molecular techniques. The objective was to develop and evaluate the contribution of massive parallel sequencing (MPS) to FFPE tissues. Methods Histopathological review of all cases was performed. Then, DNA extraction from FFPE tissues was optimized by: (1) macrodissecting the fungal-rich area on the paraffin block; (2) comparing the efficiency of two DNA extraction kits (QIAamp DNAmicro-kit, QIAGEN; Maxwell 16 LEVRNA FFPE Purification kit, Promega, optimized for RNA and DNA extraction), by comparison of Aspergillus fumigatus and Mucorale specific PCR results for 30 cases. For 124 other cases, the sensitivity of two primer pairs (ITS3/4 and MITS2A/2B) was tested for identification by Sanger sequencing and then MPS. Finally, a histomolecular comparison was performed. This work was funded by the Société de Pathologie Infectieuse de Langue Française (SPILF). Results To optimize extraction, DNA was extracted by both kits from samples of 16 mucormycoses and 14 A. fumigatus infections. PCR sensitivity was better with the QIAGEN extraction kit [100% (30/30)] compared to the Promega kit [86.7% (26/30)]. PCR amplification of fungal DNA from an additional 124 FFPE samples was performed. The primer pairs ITS3/4 and MITS2A/2B, allowed: (1) identification by Sanger sequencing-histopathological analysis in 38.7% (48/124) of the cases in total, and more specifically 33% (41/124) of cases with the ITS3/4 primers and 32.3% (40/124) of cases with the MITS2A/B primers; and (2) identification by integrated SMP-histopathological analysis in 75% (93/124) of all cases (primers ITS3/4 and MITS2A/2B), and more specifically 66.1% (82/124) for ITS3/4 and 62.1% (77/124) for MITS2A/B (both primer pairs did not detect/amplify the same fungal genera/species). The combination of all results from Sanger sequencing and MPS led to fungal identification in 75.8% (94/124) of cases. In total, the addition of NGS to Sanger sequencing increased the diagnostic proportion by 36.3% (45/124; P Conclusion The development of the fungal MPS on FFPE tissues is innovative and unprecedented for the achievement of an integrated histomolecular diagnosis in fungal pathology. It increases significantly the diagnostic proportion by 36.3%. This strategy can be used in hospitals and could improve patient management, especially when no sample is sent to the Mycology laboratory or when the culture is negative.

In this retrospective study, we report 70 cases of Epstein-Barr virus (EBV)+ diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS) among 1696 DLBCL-NOS cases diagnosed between 2006 and 2019 (prevalence of 4.1%). At diagnosis, median age was 68.5 years; 79% of the cases presented with an advanced-stage disease (III-IV), 48% with extranodal lesions, and 14% with an hemophagocytic lymphohistiocytosis (HLH) (8 at diagnosis and 1 on therapy). A total of 46 cases presented a polymorphic pattern, and 21 were monomorphic. All had a non-germinal center B phenotype, with the majority of tumor cells expressing CD30 and programmed death ligand 1 (98% and 95%, respectively). Type II and III EBV latency was seen in 88% and 12% of the cases, respectively. Patients were treated with immunochemotherapy (59%) or chemotherapy (22%), and 19% received palliative care due to advanced age and altered performance status. After a median follow-up of 48 months, progression-free survival (PFS) and overall survival (OS) at 5 years were 52.7% and 54.8%, respectively. Older age (>50 years) and HLH were associated with shorter PFS and OS in multivariate analysis (PFS: hazard ratio [HR], 14.01; 95% confidence interval [CI], 2.34-83.97; and HR, 5.78; 95% CI, 2.35-14.23; OS: HR, 12.41; 95% CI, 1.65-93.53; and HR, 6.09; 95% CI, 2.42-15.30, respectively). Finally, using a control cohort of 425 EBV− DLBCL-NOS, EBV positivity was associated with a shorter OS outcome within patients >50 years (5-year OS, 53% [95% CI, 38.2-74] vs 60.8% [95% CI, 55.4-69.3], P = .038), but not in younger patients.

Follicular lymphoma and classic Hodgkin lymphoma can be associated in composite and/or sequential lymphomas. Common IGH and BCL2 rearrangements have already been identified between both contingents of these entities, but mutation profiles have not yet been investigated. The main objective of this study was to analyze the transdifferentiation process that may occur between Hodgkin and follicular contingents in sequential and composite lymphomas to better characterize these entities. From 2004 to 2020, a retrospective multicentric study was performed, including 9 composite and 13 sequential lymphomas. Clinical data were retrospectively collected. Fluorescent in situ hybridization of BCL2 and BCL6 rearrangements, polymerase chain reaction of IGH and IGK rearrangements, next-generation sequencing of IGK rearrangement, and targeted next-generation sequencing (TNGS) on a panel of genes frequently mutated in lymphomas were performed on each contingent of composite and sequential lymphomas. For TNGS, each contingent was isolated by laser capture microdissection. Clinical presentation and evolution were more aggressive in sequential than composite lymphomas. By fluorescent in situ hybridization, common rearrangements of BCL6 and BCL2 were identified between both contingents. Similarly, a common clonal relationship was established by evaluating IGH and IGK rearrangement by polymerase chain reaction or next-generation sequencing. By TNGS, the same pathogenic variants were identified in both contingents in the following genes: CREBBP, KMT2D, BCL2, EP300, SF3B1, SOCS1, ARID1A, and BCOR. Specific pathogenic variants for each contingent were also identified: XPO1 for Hodgkin lymphoma contingent and FOXO1, TNFRSF14 for follicular lymphoma contingent. This study reinforces the hypothesis of a transdifferentiation process between Hodgkin and follicular contingent of sequential/composite lymphomas.

As the impact of targeted next-generation sequencing (TNGS) on daily diagnosis has not been evaluated, we performed TNGS (46 genes) on lymphomas of unclear subtype following expert haematopathological review. The potential impact on patient care and modifications of final diagnosis were divided into major and minor changes according to the European Society of Medical Oncology (ESMO) guidelines. Among 229 patients [19 primary central nervous system lymphomas (PCNSL), 48 large B-cell lymphomas (LBCLs), 89 small BCLs (SBCLs), seven Hodgkin lymphomas (HL), 66 T-cell lymphomas], the overall concordance rate of histological and TNGS diagnosis was 89·5%. TNGS confirmed the histological diagnosis in 144 cases (62·9%), changed the diagnosis in 24 cases (10·5%) and did not help to clarify diagnosis in 61 cases (26·7%). Modifications to the final diagnosis had a clinical impact on patient care in 8·3% of cases. Diagnostic modifications occurred in all types of lymphoma except in PCNSL and HL; the modification rate was 14·6% in SBCL and 12·5% in LBCL. While comparing informative and uninformative cases, no differences were found in terms of DNA amplification, quality or depth of sequencing and biopsy type. The present study highlights that TNGS may directly contribute to a more accurate diagnosis in difficult-to-diagnose lymphomas, thus improving the clinical management in routine practice.

p57 immunostaining is performed on hydropic products of conception to diagnose hydatidiform moles (HMs), which can progress to gestational trophoblastic neoplasia. Partial hydatidiform mole (PHM) and hydropic abortion (HA) display positive staining in stromal and cytotrophoblastic cells, whereas complete hydatidiform mole (CHM) is characterized by loss of p57 expression in both cell types. In some cases, an aberrant pattern is observed, called discordant p57 expression, with positive cytotrophoblast staining and negative stromal staining, or vice versa. The aim of this study was to describe the clinical, biological, and pathological characteristics of p57-discordant villi (p57DV) and other associated populations in cases of divergent p57 expression and to compare the evolutions of p57DV-associated and classic CHMs. Seventy cases of p57DV diagnosed by referent pathologists were divided into two groups, G1: p57DV ± non-CHM component (n = 22) and G2: p57DV + CHM component (n = 48). p57DV morphology was similar in the two groups. Observation of more than two populations and hybrid villi on p57 immunostaining were significantly more frequent in G2. The clinical, ultrasound, and biological presentations of p57DV-associated and classic CHMs were similar. The initial pathological diagnosis was more frequently incorrect, missing the CHM component, for the p57DV-associated CHMs. Molecular genotyping was informative in seven cases and identified as androgenetic/biparental mosaicism in four cases. These results show that p57DV are a diagnostic challenge for pathologists and that most are associated with a CHM component. However, the clinical management of p57DV-associated CHMs should be the same as that of classic CHMs.

Heart failure is a worldwide leading cause of death. Diet and obesity are particularly of high concern in heart disease etiology. Gravely, altered nutrition during developmental windows of vulnerability can have long-term impact on heart health; however, the underlying mechanisms are poorly understood. In the understanding of the initiation of chronic diseases related to developmental exposure to environmental challenges, deregulations in epigenetic mechanisms including micro-RNAs have been proposed as key events. In this context, we aimed at delineating the role of micro-RNAs in the programming of cardiac alterations induced by early developmental exposure to nutritional imbalance. To reach our aim, we developed a human relevant model of developmental exposure to nutritional imbalance by maternally exposing rat to high-fat diet during gestation and lactation. In this model, offspring exposed to maternal high-fat diet developed cardiac hypertrophy and increased extracellular matrix depot compared to those exposed to chow diet. Microarray approach performed on cardiac tissue allowed the identification of a micro-RNA subset which was down-regulated in high-fat diet-exposed animals and which were predicted to regulate transforming growth factor-beta (TGFβ)-mediated remodeling. As indicated by in vitro approaches and gene expression measurement in the heart of our animals, decrease in DiGeorge critical region 8 (DGCR8) expression, involved in micro-RNA biogenesis, seems to be a critical point in the alterations of the micro-RNA profile and the TGFβ-mediated remodeling induced by maternal exposure to high-fat diet. Finally, increasing DGCR8 activity and/or expression through hemin treatment in vitro revealed its potential in the rescue of the pro-fibrotic phenotype in cardiomyocytes driven by DGCR8 decrease. These findings suggest that cardiac alterations induced by maternal exposure to high-fat diet is related to abnormalities in TGFβ pathway and associated with down-regulated micro-RNA processing. Our study highlighted DGCR8 as a potential therapeutic target for heart diseases related to early exposure to dietary challenge.

Placental mesenchymal dysplasia (PMD) and complete hydatidiform mole (CHM) with a coexisting fetus are 2 rare placental abnormalities characterized by lacunar placenta and presence of an embryo on ultrasound examination. We report the case of a 34-yr-old woman referred at 32.6 weeks of gestation because of a multicystic placenta. A caesarean section was performed at 39.1 weeks of gestation giving birth to a 2905 g normal female infant. Pathological examination revealed macroscopic and microscopic morphological, and immunohistological features of PMD in the main placenta, and features of CHM in a separate placental mass. Fluorescent in situ hybridization and molecular genotyping analyses showed diandric diploidy in the CHM component and androgenetic/biparental mosaicism in the PMD component, confirming the association of PMD and CHM with a live infant. There was no progression to gestational trophoblastic neoplasia during follow-up for the mother, or any sign of Beckwith-Wiedemann syndrome or hepatic tumor in the child.

microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.

The pathogenesis of benign-looking cartilaginous tissue within the peritoneum is unknown. Chondroid metaplasia of subcoelomic mesenchyme has been suggested, as has been the case for other gynecological diseases such as endometriosis, peritoneal leiomyomatosis, or gliomatosis peritonei, but has never been proven. Chondroid nodules in the peritoneum may represent either teratomatous tissue, fetal rests from a conception product, or metaplasia of pluripotent mesenchymal cells. Herein, the unique genetic characteristics of ovarian teratomas (homozygous at many polymorphic microsatellite loci) versus normal tissues (heterozygous at the same loci) were used to investigate the origin of chondroid nodules in the peritoneum. DNA samples extracted from paraffin-embedded normal peritoneal tissue and chondroid peritoneal nodules from two patients were studied. In both cases, chondroid and normal tissue showed heterozygosity at each informative microsatellite locus on different chromosomes, with a profile similar to the mother. These results indicate that peritoneal chondroid nodules arise within the peritoneum, presumably from pluripotent mesodermal stem cells, and are not related to teratomatous proliferation, or previous pregnancy. This finding shows once again the plasticity and metaplastic potential of stem cells within the peritoneal cavity.

Introduction Aggressive B-cell lymphomas are heterogeneous in their clinical course and biological characteristics. They include diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS), high grade-B-cell lymphoma with double/triple-hit or NOS (HGBL), primary mediastinal B-cell lymphoma (PMBL). In order to better differentiate these entities, the WHO classification recommends using immunohistochemistry (IHC), FISH, targeted sequencing and gene expression profiles (GEP). However, these techniques are most often performed retrospectively in clinical trials, which is not representative of real life. In order to use these information to improve the current standard of treatment with targeted therapies adapted to lymphoma biology, we have set up a national network on behalf of the LYSA called RT3 (for Real-time tailored therapy) to demonstrate that we are able to comprehensively characterize aggressive B cell lymphomas in a clinically relevant timeline. Materials and methods Patients > 18 years of age with untreated aggressive B-cell lymphoma were included prospectively from 21 LYSA centers. FFPE specimens were analyzed and classified according to WHO classification, benefiting from an IHC profile (Hans algorithm, BCL2, MYC), FISH analysis (BCL6, BCL2, MYC break apart and MYC-IGH/IGL/IGK fusion probes), targeted NGS analysis and a targeted GEP using RT-MLPA (Bobée et al. J Mol diagn 2017). A first part of the study phase was carried out in an oligocentric manner with only 2 referral platforms for pathological analysis and molecular characterization. The second phase was the implementation of 7 RT3 platforms spread over France. The main objective was to evaluate the capacity of the network to provide an exhaustive histopathological and molecular characterization 4 days before theoretical R-CHOP21 C3 administration (within 38 days after D1 cycle 1). Results The oligocentric cohort 1 prospectively included 72 patients in 6 months: 19 had insufficient material or inappropriate diagnosis to be qualified and 53 benefited from the complete analysis on referral platforms. A complete characterization 4 days before RCHOP-C3 was provided to the clinician in 47 cases (88.7%), allowing to further implement the network with 7 platforms and 23 clinical investigation centers. 183 patients were included in the second phase in 9 months, 35 were excluded for inadequate diagnosis/material. On this population, 143 (96.6%) complete patient-reports were provided with a median time of 32 days (1-50). Finally, 201 cases were retained in the Full Analysis Set for which tumor samples fulfilled all prerequisites and diagnosis of DLBCL was confirmed by RT3 platforms. The clinical characteristics were as follows: median age of 61 y (20-92), with 45.2% of pts with aaIPI 2-3and 67.1% with Ann Arbor stage III-IV; 1L treatment consisted mainly of RCHOP (RCHOP14/21 = 136, 72.3%). 7% of patients were treated with experimental drugs. 76% presented with extranodal involvement. After pathological review, 139 (69%) patients were classified as DLBCL-NOS (41% GCB; 58% nGCB), 11 (5%) as HGBL-NOS, 8 (4%) as HGBL-DH and 18 (9%) as PMBL, 3 (1%) as EBV+ DLBCL-NOS, 9 (4%) as FL-3B, 7 (3%) transformed DLBCL, 2 (1%) plasmablastic, 1 (0.5%) unclassified, 1 (0.5%) DLBCL with IRF4 rearrangement. By immunohistochemistry, 22 cases were CD5+, 74% were BCL2+, and 53% MYC+ and 49% as double expressors. BCL2/18q21, BCL6/3q27 and MYC/8q24 breaks were observed in 14% (29/201), 25% (50/201) and 12% (25/201) of the cases respectively. MYC partner gene was available in 10/29 cases (5 MYC-IG, 5 non-IG). At the molecular level, PIM1 was the most frequently mutated gene in the entire cohort (36%). In the ABC subtype, MYD88 was mutated in 48% of cases. In the GCB cohort, SOCS1 was the most frequently mutated gene (30%). In the HGBL cohort, MYC was the most mutated gene (43%). Conclusion This study demonstrates that this RT3 LYSA network allowed a real time pathological and molecular characterization of aggressive B-cell lymphomas according to the WHO recommendations. The RT3 network provides relevant informations to the clinician before R-CHOP21 C3, that might contribute to modify the treatment eventually adding targeted therapies or tailor a second line treatment. This network should be used as a basis for future clinical trials. Inter platforms reproducibility and prognostic relevance of the RT3 project will be presented. Disclosures Haioun: Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Miltenyi: Honoraria; Servier: Honoraria; Roche: Honoraria; Celgene: Honoraria. Le Gouill:Roche Genentech, Janssen-Cilag and Abbvie, Celgene, Jazz pharmaceutical, Gilead-kite, Loxo, Daiichi-Sankyo and Servier: Honoraria; Loxo Oncology at Lilly: Consultancy. Ribrag:arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Institut Gustave Roussy: Current Employment; argenX: Current equity holder in publicly-traded company, Research Funding; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AZD: Honoraria, Other; Eisai: Honoraria; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salles:Takeda: Honoraria; Karyopharm: Honoraria; Genmab: Honoraria, Other; Debiopharm: Consultancy, Honoraria, Other: consultancy or advisory role; Autolos: Other: consultancy or advisory role; Abbvie: Other: consultancy or advisory role; Roche: Honoraria, Other: consultancy or advisory role; Novartis: Honoraria, Other: consultancy or advisory role; MorphoSys: Honoraria, Other: consultancy or advisory role; Janssen: Honoraria, Other: consultancy or advisory role; Epizyme: Honoraria, Other: consultancy or advisory role; Kite, a Gilead Company: Honoraria, Other: consultancy or advisory role ; BMS/Celgene: Honoraria, Other: consultancy or advisory role. Sujobert:Sunesis: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees. Bouabdallah:Gilead Sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Sibon:takeda france: Consultancy.

Heart diseases are a leading cause of death. While the link between early exposure to nutritional excess and heart disease risk is clear, the molecular mechanisms involved are poorly understood. In the developmental programming field, increasing evidence is pointing out the critical role of epigenetic mechanisms. Among them, polycomb repressive complex 2 (PRC2) and DNA methylation play a critical role in heart development and pathogenesis. In this context, we aimed at evaluating the role of these epigenetic marks in the long-term cardiac alterations induced by early dietary challenge. Using a model of rats exposed to maternal high-fat diet during gestation and lactation, we evaluated cardiac alterations at adulthood. Expression levels of PRC2 components, its histone marks di- and trimethylated histone H3 (H3K27me2/3), associated histone mark (ubiquitinated histone H2A, H2AK119ub1) and target genes were measured by Western blot. Global DNA methylation level and DNA methyl transferase 3B (DNMT3B) protein levels were measured. Maternal high-fat diet decreased H3K27me3, H2Ak119ub1 and DNA methylation levels, down-regulated the enhancer of zeste homolog 2 (EZH2), and DNMT3B expression. The levels of the target genes, isl lim homeobox 1 (Isl1), six homeobox 1 (Six1) and mads box transcription enhancer factor 2, polypeptide C (Mef2c), involved in cardiac pathogenesis were up regulated. Overall, our data suggest that the programming of cardiac alterations by maternal exposure to high-fat diet involves the derepression of pro-fibrotic and pro-hypertrophic genes through the induction of EZH2 and DNMT3B deficiency.

Human and wildlife exposure to chemicals is thought to be extensive and particularly to endocrine-disrupting chemicals (EDCs) suspected to alter male reproductive tract. When the exposure occurs during perinatal period (fetal, neonatal periods or puberty) the reproductive health alterations are irreversible suggesting a developmental origin to male infertility. This concept is supported by numerous epidemiologic and experimental studies. This review summarizes the data concerning the epigenetic mechanisms (DNA methylation, chromatin remodelling, small-non coding RNAs) involved in developmentally-induced male infertility. These data open potentially to new diagnosis tools and new trails to assessment of EDCs risks.

Paternal exposure to environmental challenges plays a critical role in the offspring's future health and the transmission of acquired traits through generations. This review summarizes our current knowledge in the new field of epigenomic paternal transmission of health and disease. Epidemiological studies identified that paternal ageing or challenges (imbalanced diets, stress, toxicants, cigarette smoke, alcohol) increased the risk of offspring to develop diseases such as cancer, metabolic, cardiovascular, and neurological diseases. These data were confirmed and deepened in animal models of exposure to challenges including low-protein, low-folate, high-fat diets, exposure to chemicals such as pesticides and herbicides. Even though some toxicants have mutagenic effect on sperm DNA, changes in sperm epigenome seem to be a common thread between different types of challenges. Indeed, epigenetic changes (DNA methylation, chromatin remodeling, small non-coding RNA) in sperm are described as new mechanisms of intergenerational transmission as demonstrated for dioxin, for example. Those epimutations induce dysregulation in genes expression involved in key cellular pathways such as reactive oxygen species and genome stability regulation, in brain-derived neurotrophic factor, calcium and glucocorticoid signaling, and in lipid and glucose metabolism, leading to diseases in offspring. Finally, since each type of environmental challenges has its own signature by inducing epimutations at specific genomic loci, the sperm epigenome might be used as a biomarker in toxicological and risk assessments.

Epidemiological and experimental observations tend to prove that environment, lifestyle or nutritional challenges influence heart functions together with genetic factors. Furthermore, when occurring during sensitive windows of heart development, these environmental challenges can induce an ‘altered programming’ of heart development and shape the future heart disease risk. In the etiology of heart diseases driven by environmental challenges, epigenetics has been highlighted as an underlying mechanism, constituting a bridge between environment and heart health. In particular, micro-RNAs which are involved in each step of heart development and functions seem to play a crucial role in the unfavorable programming of heart diseases. This review describes the latest advances in micro-RNA research in heart diseases driven by early exposure to challenges and discusses the use of micro-RNAs as potential targets in the reversal of the pathophysiology.

International audience; Background and aims: The prevalence of obesity is increasing worldwide at an alarming rate. Altered early nutrition, in particular postnatal overfeeding (PNOF), is a risk factor for impaired cardiac function in adulthood. In the understanding of the initiation or progression of heart diseases, NLRP3 inflammasome and non-coding RNAs have been proposed as key players. In this context, the aim of this study was to decipher the role of NLRP3 inflammasome and its post transcriptional control by micro-RNAs in the regulation of cardiac metabolic function induced by PNOF in mice. Methods and results: Based on a model of mice exposed to PNOF through litter size reduction, we observed increased cardiac protein expression levels of NLRP3 and ETS-1 associated with alterations in insulin signaling. Additionally, miR-193b levels were down-regulated in the adult hearts of overfed animals. In a cardiomyocyte cell line, transfection with miR-193b induced down-regulation of ETS-1 and NLRP3 and improved insulin signaling. Conclusions: These findings suggest that the miR-193b could be involved in cardiac phenotypic changes observed in adulthood induced by PNOF likely through the regulation of ETS-1 and NLRP3 expression, and through this of insulin signaling.

Study question Do TNF-308 and -238 polymorphisms impact the embryo implantation rate after in vitro fertilization (IVF) in women without female infertility factor? Summary answer The presence of the TNF-308A allele is associated with high implantation and multiple pregnancy rates in women without known infertility factors after ovarian hyperstimulation with exogenous FSH. What is already known Multiple pregnancies are frequent after the use of Assisted Reproductive Technologies. Single embryo transfer (SET) has been proposed as a simple way to prevent these risks. However, the extension of SET indications to patients not selected based on specific criteria is controversial because of reduced pregnancy rates. To date, the predictive value of the parameters used for SET (age, gynecological history of the patient and uterine characteristics) allows a pregnancy rate of ~30%. Study design, size, duration The potential predictive value of TNF polymorphisms (-308, rs1800629 and -238, rs361525) on implantation rate was evaluated in 424 women requiring IVF due to male fertility factors. This cohort retrospective study was conducted over 4 years in University-affiliated hospitals. Participants, setting, methods The entire patient group included 424 women undergoing intracytoplasmic sperm injection (ICSI) due to male fertility factors without the contribution of any female factor. From among this group, a selected patient group included 120 women with a normal karyotype, age under 38 years, serum follicle-stimulating hormone (Day-3 FSH) levels below 10 IU/l, a long agonist desensitization protocol associated with recombinant FSH treatment and a Caucasian background. Main results and the role of chance The TNF-238 polymorphism was not associated with implantation rate. In contrast, the presence of the TNF-308A allele was associated with increased Day 3-E2 levels as well as higher implantation and multiple pregnancy rates after fresh embryo transfer in women from the entire and selected patient groups. Moreover, in the selected patient group, the presence of the TNF-308A allele was also associated with a decrease in the miscarriage rate. The benefit of the TNF-308A allele in predicting implantation rates was not observed after the use of frozen embryos. Limitations, reasons for caution Future studies are needed to evaluate whether the TNF-308A allele might also be a biomarker in women with infertility factors. Wider implications of the finding The TNF-308A allele may represent a good candidate for a potential predictive, non-invasive biomarker in the SET strategy. However, its impact should be evaluated in prospective studies. Study funding/competing interest This study was conducted with financial support from the French Institute for Health and Medical Research (INSERM), Organon France for a FARO (Fond d'Aide a la Recherche Organon) fellowship (to V.T.) and CHU Nice PHRC (PHRC 09-279).There are no competing interests.

International audience; The DICER1 gene encodes an endoribonuclease involved in the production of mature microRNAs which regulates gene expression through several mechanisms. Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Pleuropulmonary blastoma is the most frequent lesion seen in this syndrome. Thyroid abnormalities are also a common finding, essentially concerning multinodular goiter. However, differentiated thyroid carcinoma is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 RNase IIIb catalytic domain in several tumor types, including ovarian Sertoli-Leydig cell tumors. We report two cases of differentiated thyroid carcinoma associated with ovarian Sertoli-Leydig cell tumor and with a heterozygous DICER1 gene mutation, occurring in two unrelated young girls without pleuropulmonary blastoma. Both thyroid carcinomas showed an E1813 mutation in exon 25 while the ovarian tumors harboured a somatic mutation in E1705 in exon 24 and a D1709 mutation in exon 25. Our observations confirm that the occurrence of an ovarian Sertoli-Leydig cell tumor with a thyroid carcinoma is highly suggestive of a DICER1 syndrome. We contend that the possibility of a relationship between sporadic thyroid carcinoma in young patients and somatic DICER1 gene mutation needs further investigation

Human and wildlife exposure to chemicals is thought to be extensive and particularly to endocrine-disrupting chemicals (EDCs) suspected to alter male reproductive tract. When the exposure occurs during perinatal period (fetal, neonatal periods or puberty) the reproductive health alterations are irreversible suggesting a developmental origin to male infertility. This concept is supported by numerous epidemiologic and experimental studies. This review summarizes the data concerning the epigenetic mechanisms (DNA methylation, chromatin remodelling, small-non coding RNAs) involved in developmentally-induced male infertility. These data open potentially to new diagnosis tools and new trails to assessment of EDCs risks.

A differential responsiveness of patients to ionizing radiation is observed after preoperative radiotherapy for rectal adenocarcinoma that might be related, in part, to an apoptosis defect. To establish if proteins of the apoptotic cascades [pro-apoptotic: active caspase 3, 8, and 9 and DIABLO (direct inhibitor of apoptosis-binding protein with low pI); anti-apoptotic: XIAP (X-linked inhibitor of apoptosis)] are involved, we analyzed their profile in radioresistant (SW480) and radiosensitive (SW48) human colorectal cell lines. We demonstrated that, after irradiation, the SW48 cells increased the expression of the pro-apoptotic proteins, whereas the SW480 cells increased the expression of the anti-apoptotic protein XIAP. Moreover, XIAP knockdown in SW480 cells enhanced the basal and radiation-induced apoptotic index; the propensity of the SW480 cells to undergo apoptosis after radiation was higher compared with SW48 cells. In a translational study of 38 patients with rectal carcinoma, we analyzed the apoptotic profile for tumor and noncancerous tissue for each biopsy specimen using IHC. According to their response to preoperative radiotherapy, patients were classified into two groups: responsive and nonresponsive. Although no difference in expression of caspase 3, 8, or 9 was observed in the tumor/normal tissue ratio between responsive and nonresponsive patients, the ratio decreased for DIABLO and increased for XIAP. In conclusion, inhibition of XIAP rescues cellular radiosensitivity and both DIABLO and XIAP might be potential predictive markers of radiation responsiveness in rectal adenocarcinoma.

BACKGROUND One of the most well-documented cytokines suspected as a hazard to male fertility is tumor necrosis factor-alpha (TNFalpha). Genetic factors such as single-nucleotide polymorphisms (SNPs) in the TNF gene cluster impact TNFalpha levels. Our objective was to establish the potential involvement of -308 TNF SNP in male infertility risk. METHODS In 684 infertile male patients undergoing an intracytoplasmic sperm injection procedure, we used allele-specific polymerase chain reaction (PCR) and PCR-RFLP to investigate the distribution of the guanine (G)-to-adenosine (A) substitution at position -308 in the promoter region of the TNFalpha gene. RESULTS An increased frequency of the -308 TNFalpha A allele was found in patients with low sperm count of testicular origin [P = 0.002; odds ratio (OR) = 2.93] or with normal production count but altered sperm motility (P = 0.003; OR = 2.32), compared with a patient group with normal sperm count and quality (morphology and motility). In patients with low sperm count exhibiting TNFalpha A allele, compared with those with G allele, an alteration in hormonal balance was observed with increased inhibin B levels and subsequent reduced FSH plasma levels, leading to an FSH/inhibin B ratio roughly half as high (from 0.07 +/- 0.01 in TNFA versus 0.13 +/- 0.02 in TNFG allele groups, P < 0.0001). CONCLUSION As the -308 TNFalpha A allele has been associated with an increased expression/production of TNFalpha, the potential use of therapies based on inhibition of TNFalpha activities could represent possible therapeutic opportunities for patients with low sperm count (i.e. primary testicular dysfunction) or with altered sperm motility.

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of AR-containing regulatory complexes and that high levels of NPM might alter AR functions in PCa.

Fetal androgen disruption, induced by the administration of anti-androgen flutamide (0.4, 2, and 10 mg/kg day) causes a long-term apoptosis in testicular germ cells in adult male rat offspring. One of the questions raised by this observation is the role of the Sertoli cells in the adult germ cell apoptotic process. It is shown here that Sertoli cells originating from 15-day-old rats treated in utero with the anti-androgen (10 mg/kg d) did no longer protect adult germ cells against apoptosis. Indeed, untreated spermatocytes or spermatids exhibited increased (Pin utero to the anti-androgen. This alteration of Sertoli cell functions was not due to modifications in the androgen signal in the adult (90-day-old) animals, since plasma testosterone and estradiol, androgen receptor expression, and androgen-targeted cell number (e.g., Sertoli cells in the seminiferous tubules) were not affected by the fetal androgen disruption. In contrast, this inability of Sertoli cells to protect germ cells against apoptosis could be accounted for by the potential failure of Sertoli cell functions. Indeed, adult testes exposed in utero to anti-androgens displayed decreased levels of several genes mainly expressed in adult Sertoli cells (anti-Mullerian hormone receptor type II (AMHR2), Cox-1, cyclin D2, cathepsin L, and GSTα). In conclusion, fetal androgen disruption may induce alterations of Sertoli cell activity probably related to Sertoli cell maturation, which potentially leads to increased adult germ cell apoptosis.

The purpose here was to identify whether ionizing radiation and oxaliplatin triggered testicular germ cells apoptosis through different executionary pathways.Adult male mice are treated with oxaliplatin (0.5 mg/kg, Ox) 4 h before being locally irradiated (0.5 Gy, IR, considered as time 0 h).The number of apoptotic germ cells was significantly higher for IR (p0.008), Ox (p0.0001) and Ox + IR (p0.0001) groups compared to the untreated mice group. Similarly, the different treatments induced an increase of p53 expression. Downstream p53, IR and Ox used different pathways. Indeed, IR increased effector caspase-3 expression in terms of mRNA (p0.002), pro-enzyme p0.0001) and active (3.7-fold, p0.003) protein levels but not the inhibitors of apoptosis proteins (IAP) including cIAP1, cIAP2 and XIAP. In contrast, while oxaliplatin treatment had no apparent effect on caspase-3 expression, it significantly decreased the cIAP1 (p0.005), cIAP2 (p0.008) and XIAP (p0.02) proteins levels. Finally, the combination of both treatments decreased IAP expression but did not modify caspase-3 levels while it increased the AIF (apoptosis-inducing factor) protein levels (5.5-fold, p0.003). No modification of AIF levels was observed with OX or IR alone.Together, these results indicate that the platinum analogue oxaliplatin and the ionizing radiations trigger apoptosis in the testicular germ cells, probably through different pathways.

Several epidemiologic studies have demonstrated during the last 50 years an increased incidence in testis cancer, male genital tract malformations (cryptorchidism and hypospadias) and a decrease in sperm quality in men. These three pathologies seem to be linked and to belong to the testicular dysgenesis syndrome (TDS). It was suggested that TDS is a consequence of intra-uterine exposure to environmental compounds that disrupt the metabolism of native hormones. Such substances are so called endocrine disruptors (EDs). EDs are present in our daily environment such as food and water (through the use of pesticides), cosmetics, house-care products etc. Experimental models have been carried out to (i) establish a link between EDs exposure and SDT and (ii) identify the mechanisms that are involved in. After a brief definition of EDs and having underlined the importance of the window of exposure to EDs, several mechanisms will be described such as (i) intergenerational transmission (epigenetic), (ii) programmed cell death of testicular cells, (iii) modification of the androgenic signal and (iv) role of the germ cells-nourishing cells. To conclude, we will try to propose some biomarkers that would be useful to identify the potential link between fetal exposure to anti-androgenic EDs and male testicular pathology.

In utero exposure to chemicals with antiandrogen activity induces undescended testis, hypospadias, and sub- or infertility. The hypospermatogenesis observed in the adult rat testis exposed in utero to the antiandrogen flutamide has been reported to be a result of a long-term apoptotic cell death process in mature germ cells. However, little if anything is known about the upstream signaling mechanisms controlling this apoptosis. In the present study, we have investigated the possibility that the TGF-beta signaling pathway may be at play in this control of the apoptotic germ cell death process. By using a model of adult rat exposed in utero to 0, 0.4, 2, or 10 mg/kg.d flutamide, we observed that pro-TGF-beta signaling members, such as the three isoforms of TGF-beta ligands (TGF-beta1-3), the two TGF-beta receptors (TGF-betaRI and -RII) and the R-Smads Smad 1, Smad 2, Smad 3, and Smad 5 were inhibited at the mRNA and protein levels, whereas the anti-TGF-beta signaling member Smad 7 was overexpressed. Furthermore, we report that the overexpression of Smad 7 mRNA could induce an activation of c-Jun N-terminal kinase, because of the observed c-Jun overexpression, activation, and nuclear translocation leading to an increase in the transcription of the proapoptotic factor Fas-L. Together, the alterations of TGF-beta signaling may represent upstream mechanisms underlying the adult germ cell apoptotic process evidenced in adult rat testis exposed in utero to antiandrogenic compounds such as flutamide.

The involvement of mutated androgen receptors (mut-AR) in the actions of estrogens in prostate cancer cells is controversial. This work was designed to determine the role of such receptors in the growth inhibition by estradiol (E2) and androgens of the MOP cell line, a derivative of the LNCaP cell line. Diethylstilbestrol (DES) was used as a “tool”. E2 like DHT and R1881 inhibits MOP cell proliferation while DES does not. E2 and R1881 down regulate mut-AR mRNA, DES does not. E2 enhances mut-AR transcriptional activity less efficiently than R1881 while DES does not. E2 and R1881 up regulate PSA secretion in a dose-dependent manner, DES does it marginally at 10 −6 M. MOP cells express low amounts of ERα and ERβ mRNA but neither DES nor E2 and R1881 do enhance ER transcriptional activity. DES and E2 bind to mut-AR with relative binding affinities which are respectively 1/175 and 1/10 that of DHT. The E2 and androgen-repressed proliferation is prevented by DES and by the anti-androgen bicalutamide. In LNCaP cells, DES prevents the androgen-enhanced proliferation. These results strongly suggest that: (a) the putative endogenous ERs are biologically inactive in MOP cells, (b) the E2-repressed proliferation results from hormone binding to mut-AR and, (c) DES is an anti-androgen in mut-AR expressing cell line.

During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFalpha and TGFbeta also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFbeta and TNFalpha, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell-germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.

Although it is established that in utero exposure to antiandrogenic compounds such as flutamide induces hypospermatogenesis in adult male rat offspring, the cellular and molecular mechanisms remain to be investigated. By using adult rats exposed in utero to flutamide (0.4, 2, 10 mg/kg.d) as a model, we show that the hypospermatogenesis could be related to a chronic apoptotic cell death process associated with a long-term increase in caspase-3 and -6 expression and activation in germ cells. The number of apoptotic (terminal deoxynucleotidyl transferase-mediated deoxyuridine positive) adult germ cells was dependent on the dose of flutamide. The apoptotic germ cell death process could be related to an increased expression and activation of effector caspases-3 and -6. Procaspases-3 and -6 were immunodetected in germ cells from both untreated or flutamide-treated rats, whereas cleaved active caspase-3 was detected exclusively in germ cells from adult rat exposed in utero to flutamide. Exposure to the antiandrogen increased in a dose-dependent manner as caspase-3 and -6 mRNA (in RT-PCR approaches) as well as procaspase-3 and -6 protein (in Western blotting analyses) levels in the adult rat testis. Flutamide also activates procaspases. Indeed, whereas cleaved active caspase-3 and -6 proteins were absent in control animals, they were detected in adult rat testes exposed in utero to flutamide. Our results show that whereas the apoptotic germ cell death process associated with the increased caspase expression and activation in adult rat germ cells was chronic and nonreversible when exposure to flutamide occurred in utero, it was transient when such an exposure occurred during adulthood. Indeed, although an increase in caspase-3 and -6 mRNA and procaspase-3 and -6 protein levels was observed in germ cells after 3 d of exposure to flutamide, 1-2 wk after the cessation of the antiandrogen exposure, the caspase mRNA and procaspase protein levels were back to control. Active cleaved caspase-3 and -6 protein appeared following the exposure to the antiandrogen, whereas they disappeared at cessation of exposure to flutamide. In summary, the present findings indicate that in utero exposure to the antiandrogen induced in the adult rat testes a chronic apoptotic germ cell death associated with a long-term increase in the expression and activation in germ cells of caspases-3 and -6, two key components in the death machinery.

The molecular basis of testicular germ cell tumourigenesis are not well elucidated. Growth factors regulate cell growth, differentiation and apoptosis. Major families of growth factors are present in the male gonad from early fetal development to adult life. They are involved in germ cell proliferation and differentiation. Growth signalling pathways suffer deregulation in many human malignancies. Given the importance of growth signals in normal testicular development and their acquired deregulation in most human cancers, growth factors and signalling molecules that have been implicated in the genesis of testicular germ cell tumours, are reviewed. We detected a somatic mutation of SMAD4 gene, responsible for loss of protein function in seminomas. This mutational inactivation may affect the activity of several members of TGFbeta superfamily (TGFbeta, activin, inhibin, BMP). VEGF expression has been shown to predict metastasis in seminomas. A significant association of HST-1 expression, a member of fibroblast growth factors, with the nonseminomatous phenotype and with tumour stage has been described. In contrast, C-KIT is expressed by seminomas only, from the preinvasive stage. Despite intense expression in almost all seminomas, activating mutation of C-KIT gene is seldom reported. Recently, the first animal model of classical testicular seminoma has been identified in transgenic mouse overexpressing GDNF. RET (GDNF receptor) expression is demonstrated in human seminomas, and not in nonseminomatous tumours. However, the exact molecular alterations of GDNF/RET/GFRalpha1 complex in germ cell tumours are not known. Finally, beside growth factors, other signalling molecules such as peptide hormones may be involved in testicular carcinogenesis. We have demonstrated a specific pattern of somatostatin receptors expression in each type of testicular germ cell tumours, with a loss of sst3 and sst4 in seminomas and loss of sst4 and expression of sst1 in nonseminomas only. These data suggest an antiproliferative action of somatostatin in testicular cancers. In summary, many growth factors and signalling molecules seem to represent specific markers for different histological types of germ cell tumours (seminomas versus nonseminomas) and may play a role in the differentiation of germ cell tumours. Despite a complex signalling pathway involved in the physiological functions of male gonad, little is known about the implication of this signalling network in testicular malignancies. From a practical stand-point, further studies on the role of growth factors in human germ cell tumours may offer a new therapeutical perspective with the development of specific pharmacological signalling modulators that could be used as therapeutic agents.

Objet Les maladies non transmissibles (NMT, syndrome metabolique, diabete, obesite, certains cancers, infertilite) representent un probleme majeur de sante publique. En 1989, D. Barker [1] a montre que le syndrome metabolique de l’adulte etait lie aux evenements precoces de la vie (malnutrition pendant la periode perinatale). Plus largement, ces dernieres annees ont vu l’emergence du concept de programmation perinatale de certaines MNT. L’infertilite masculine serait, dans certains cas, liee a une exposition a des molecules perturbateurs endocriniens (PE)œstrogeniques (pesticides, plastifiants). Le but de notre etude etait d’identifier des mecanismes d’action des PE. Methode Nous avons developpe un modele de rats mâles exposes entre le 1e et le 5e jour apres la naissance a un PE l’estradiol benzoate. Resultats Les rats adultes presentent une hypospermatogenese/infertilite dependante de la dose administree. Ce phenotype est lie a une apoptose accrue dans les cellules germinales meiotiques. Nous montrons que ce phenotype est lie a une alteration de l’expression d’effecteurs epigenetiques avec une augmentation d’ARNs non codants, miR-29 et 18a, qui entraine une diminution de l’expression leurs cibles les DNA methyltransferases et les proteines de choc thermiques (HSP70). La diminution de ces proteines entraine une diminution d’expression des proteines anti-apoptotiques et une augmentation des pro-apoptotiques et l’apoptose des cellules germinales adultes. L’alteration des miRs est observee aussi bien au niveau tissulaire que plasmatique. Conclusion Notre travail montre qu’une exposition perinatale a un PE estrogenique programme une infertilite chez l’adulte via une alteration d’une voie de signalisation epigenetique.

MiRNAs (microRNAs) are single-stranded non-coding RNAs of approximately 21–23 nucleotides in length whose main function is to inhibit gene expression by interfering with mRNA processes. MicroRNAs suppress gene expression by affecting mRNA (messenger RNAs) stability, targeting the mRNA for degradation, or both. In this review, we have examined how microRNA expression could be altered following exposure to chemicals and how they could represent appropriate tissue and more interestingly circulating biomarkers. Among the key questions before using the microRNA for evaluation of risk toxicity, it remains still to clarify how they could be causally involved in the adverse effects and how stable their changes are.

The aim of this study was to investigate DNA mismatch repair deficiency in male germ cell tumours. We analysed the expression of two mismatch repair proteins, human mutL homologue 1 (hMLH1) and human mutS homologue 2 (hMSH2), and evaluated the frequency of microsatellite instability with 10 mononucleotide and two dinucleotide repeat sequences, in 39 paired tumour/normal DNA samples obtained from 17 testicular and two mediastinal germ cell tumours. In all 19 cases, hMLH1 and hMSH2 both showed nuclear immunolocalization in invasive and testicular in-situ tumours. In non-neoplastic seminiferous tubules, hMLH1 was expressed only in premeiotic germ cells, while hMSH2 was seen in all stages of spermatogenesis. Genetic analysis of dinucleotide markers revealed loss of heterozygosity in one of two testicular yolk sac tumours at D18S58 and an allelic shift at D2S123 in two of three testicular embryonal carcinomas, while none of the 12 seminomas exhibited a genetic abnormality at these loci. No abnormalities were demonstrated with the 10 mononucleotide markers. The two mediastinal germ cell tumours showed no genetic instability or allelic loss with all 12 markers. We suggest that genetic alterations as assessed by microsatellite analysis in germ cell tumours may reflect tissue maturation and phenotypic differentiation rather than tumour progression. In addition, we suggest that hMLH1 and hMSH2 genes may not be implicated in the genesis of germ cell tumours.

Porcine Leydig cells in primary cultures are resistant to tumor necrosis factor alpha (TNFα) cytotoxicity. Here we report that these cells can be rendered sensitive to TNFα killing by treatment with the translational inhibitor cycloheximide, suggesting the existence of proteins that can suppress the death stimulus induced by the cytokine. In search of these cytoprotective proteins, we focused on the constituents of the mitochondrial permeability transition pore (PT pore), whose opening has been shown to play a critical role in the TNFα-mediated death pathway. We found that TNFα up-regulated mRNA and protein expression of the mitochondrial peripheral benzodiazepine receptor (PBR), an outer membrane-derived constituent of the pore. A strong correlation was established between the resistance of the cells to TNFα killing and the density of PBR-binding sites. Concomitantly, TNFα down-regulated Bcl-2 mRNA and protein expression. As Bcl-2 has been shown to be an endogenous inhibitor of the PT pore, we hypothesize that the TNFα-induced up-regulation of PBR expression may compensate for the decrease in Bcl-2 levels to prevent the opening of the PT pore.

On estime a l’heure actuelle qu’environ 10% des couples dans le monde sont concernes par des problemes de procreation. Dans au moins la moitie des cas d’infertilite, c’est le partenaire masculin qui est en cause. Les infertilites masculines ont soit une origine non obstructive (l’anomalie est testiculaire ce sont les infertilites dites secretoires) ou obstructive (l’anomalie est observee sur les voies excretrices). Les infertilites secretoires touchent environ 2% des hommes dans le monde. Ces infertilites sont dues a un deficit primaire dans la production de spermatozoides. Si pour certaines de ces affections une origine genetique, physique, chimiotherapeutique ou infectieuse a ete identifiee, pour plus de la moitie d’entre elles, la cause reste encore inconnue. Au cours de ces dernieres annees, les travaux de recherche fondamentale ont apporte un eclairage nouveau et suggerent qu’une atteinte des systemes de signalisation cellulaire pourrait etre une cause potentielle de certaines infertilites secretoires idiopathiques. Ainsi, les experiences de transgenese ont montre que l’absence d’expression ou la surexpression de certaines molecules de signalisation provoque une alteration de la spermatogenese chez l’animal. C’est le cas pour les systemes TGFβ et SCF/c-kit, par exemple. Les donnees experimentalesin vivo mais surtoutin vitro, ont permis de mieux comprendre les mecanismes par lesquels certaines molecules de signalisation intragonadiques regulent les fonctions testiculaires en interaction avec les hormones (LH/testosterone, FSH). Ces facteurs gonadiques joueraient un role important pendant la vie fœtale, probablement en interaction avec les genes du developpement gonadique, mais aussi a l’âge adulte ou ces facteurs reguleraient la spermatogenese en tant que relais des systemes endocriniens classiques. Des etudes recentes suggerent, deja, que les facteurs locaux pourraient etre a l’origine de certaines pathologies testiculaires chez l’homme. Il est a parier que les travaux futurs vont confirmer le role joue par ces molecules en pathologie testiculaire humaine.

The proliferation and differentiation of testicular progenitor stem cells into highly specialized germ cells (spermatozoa) are largely controlled by the hormonally (FSH and testosterone) regulated adjacent supporting Sertoli cells. However, the factors involved in this control remain largely unknown. In the present study, the technique of differential display PCR was used to identify target transcripts to FSH action in cultured murine Sertoli cells. Among these target transcripts, we identified the oligodendrocyte-specific protein (OSP), also known as claudin 11, which had recently been shown to play a key role in the formation of the hematotesticular barrier. Our data show that the testicular expression of OSP is dependent upon male gonad development and systemic and local signaling molecules. Indeed, OSP is expressed early in fetal development in Sertoli cells, immediately after the peak of SRY (sex-determining region, Y gene) expression, but just before that of the anti-Mullerian hormone. Postnatally, ...

One of the major unresolved questions with male infertility is the identification of the molecular origin of a great majority of the spermatogenetic arrests currently diagnosed as idiopathic male infertility. During the past years, several families of regulating factors have been implicated in spermatogenesis defects observed essentially in animal models. Among these factors are signalling molecules, and particularly the stem cell factor (SCF)/c-kit system. The SCF and its receptor c-kit are an appropriate example to illustrate the role of signalling molecules in the physiology and pathology of spermatogenesis. The SCF/c-kit regulates primordial germ cell migration, proliferation and apoptosis during fetal gonadal development. The SCF/c-kit also regulates spermatogonia proliferation in the adult animal. In mutant mice, abnormalities of the SCF/c-kit gene expression, such as gene deletion, point mutation, alternative splicing defect, lead to different types of spermatogenesis alterations (e.g. decrease in primordial germ cell migration, decrease in spermatogonia proliferation). More recently, defects in SCF/c-kit gene expression have also been shown in human testicular dysfunctions. Indeed, a reduction in SCF/c-kit expression has been evidenced in oligozoospermia/azoospermia associated with an increase in the germ cell apoptosis process. In addition, c-kit seems to be a good marker of seminoma testicular tumours. This review reports a large number of data--obtained essentially in animal models--that suggest an important role for the SCF/c-kit system in spermatogenesis and, as a corollary, its potential involvement in spermatogenic defects.