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Recent advancements in reproductive medicine have guided novel strategies for addressing male infertility, particularly in cases of non-obstructive azoospermia (NOA). Two prominent invasive interventions, namely testicular sperm extraction (TESE) and microdissection TESE (micro-TESE), have emerged as key techniques to retrieve gametes for assisted reproduction technologies (ART). Both heterogeneity and complexity of NOA pose a multifaceted challenge to clinicians, as the invasiveness of these procedures and their unpredictable success underscore the need for more precise guidance. Seminal plasma can be aptly regarded as a liquid biopsy of the male reproductive tract, encompassing secretions from the testes, epididymides, seminal vesicles, bulbourethral glands, and prostate. This fluid harbors a variety of cell-free nucleic acids, microvesicles, proteins, and metabolites intricately linked to gonadal activity. However, despite numerous investigations exploring potential biomarkers from seminal fluid, their widespread inclusion into the clinical practice remains limited. This could be partially due to the complex interplay of diverse clinical and genetic factors inherent to NOA that likely contributes to the absence of definitive biomarkers for residual spermatogenesis. It is conceivable that the integration of clinical data with biomarkers could increase the potential in predicting surgical procedure outcomes and their choice in NOA cases. This comprehensive review addresses the challenge of sperm retrieval in NOA through non-invasive biomarkers. Moreover, we delve into promising perspectives, elucidating innovative approaches grounded in multi-omics methodologies, including genomics, transcriptomics and proteomics. These cutting-edge techniques, combined with the clinical and genetics features of patients, could improve the use of biomarkers in personalized medical approaches, patient counseling, and the decision-making continuum. Finally, Artificial intelligence (AI) holds significant potential in the realm of combining biomarkers and clinical data, also in the context of identifying non-invasive biomarkers for sperm retrieval. [ABSTRACT FROM AUTHOR]

Beckwith–Wiedemann Syndrome (BWS) is an imprinting disorder characterized by overgrowth, stemming from various genetic and epigenetic changes. This study delves into the role of IGF2 upregulation in BWS, focusing on insulin-like growth factor pathways, which are poorly known in this syndrome. We examined the IGF2R, the primary receptor of IGF2, WNT, and autophagy/lysosomal pathways in BWS patient-derived lymphoblastoid cell lines, showing different genetic and epigenetic defects. The findings reveal a decreased expression and mislocalization of IGF2R protein, suggesting receptor dysfunction. Additionally, our results point to a dysregulation in the AKT/GSK-3/mTOR pathway, along with imbalances in autophagy and the WNT pathway. In conclusion, BWS cells, regardless of the genetic/epigenetic profiles, are characterized by alteration of the IGF2R pathway that is associated with the perturbation of the autophagy and lysosome processes. These alterations seem to be a key point of the molecular pathogenesis of BWS and potentially contribute to BWS's characteristic overgrowth and cancer susceptibility. Our study also uncovers alterations in the WNT pathway across all BWS cell lines, consistent with its role in growth regulation and cancer development. [ABSTRACT FROM AUTHOR]

Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient's life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner. [ABSTRACT FROM AUTHOR]

Background: Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder caused by defects at the 11p15.5 imprinted region. Many cases of female monozygotic (MZ) twins discordant for BWS have been reported, but no definitive conclusions have been drawn regarding the link between epigenetic defects, twinning process, and gender. Here, we report a comprehensive characterization and follow‐up of female MZ twins discordant for BWS. Methods: Methylation pattern at 11p15.5 and multilocus methylation disturbance (MLID) profiling were performed by pyrosequencing and MassARRAY in placental/umbilical cord samples and postnatal tissues. Whole‐exome sequencing was carried out to identify MLID causative mutations. X‐chromosome inactivation (XCI) was determined by HUMARA test. Results: Both twins share KCNQ1OT1:TSS‐DMR loss of methylation (LOM) and MLID in blood and the epigenetic defect remained stable in the healthy twin over time. KCNQ1OT1:TSS‐DMRLOM was nonhomogeneously distributed in placental samples and the twins showed the same severely skewed XCI pattern. No MLID‐causative mutations were identified. Conclusion: This is the first report on BWS‐discordant twins with methylation analyses extended to extraembryonic tissues. The results suggest that caution is required when attempting prenatal diagnosis in similar cases. Although the causative mechanism underlying LOM remains undiscovered, the XCI pattern and mosaic LOM suggest that both twinning and LOM/MLID occurred after XCI commitment. [ABSTRACT FROM AUTHOR]

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting-related disorders associated with genetic/epigenetic alterations of the 11p15.5 region, which harbours two clusters of imprinted genes (IGs). 11p15.5 IGs are regulated by the methylation status of imprinting control regions ICR1 and ICR2. 3D chromatin structure is thought to play a pivotal role in gene expression control; however, chromatin architecture models are still poorly defined in most cases, particularly for IGs. Our study aimed at elucidating 11p15.5 3D structure, via 3C and 3D FISH analyses of cell lines derived from healthy, BWS or SRS children. We found that, in healthy cells, IGF2/H19 and CDKN1C/KCNQ1OT1 domains fold in complex chromatin conformations, that facilitate the control of IGs mediated by distant enhancers. In patient-derived cell lines, we observed a profound impairment of such a chromatin architecture. Specifically, we identified a cross-talk between IGF2/H19 and CDKN1C/KCNQ1OT1 domains, consisting in in cis, monoallelic interactions, that are present in healthy cells but lost in patient cell lines: an inter-domain association that sees ICR2 move close to IGF2 on one allele, and to H19 on the other. Moreover, an intra-domain association within the CDKN1C/KCNQ1OT1 locus seems to be crucial for maintaining the 3D organization of the region. [ABSTRACT FROM AUTHOR]

X‐linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X‐linked heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3′‐phosphate, 5′‐phosphosulfate to the sixth position of the N‐sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in‐silico tools. An in‐vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here. [ABSTRACT FROM AUTHOR]

Background: Omphalocele is a congenital midline ventral body wall defect that can exist as isolated malformation or as part of a syndrome. It can be considered one of the major and most frequent clinical manifestation of Beckwith-Wiedemann Syndrome (BWS) in case of loss of methylation at KCNQ1OT1: Transcription Star Site-Differentially Methylated Region (TSS-DMR) or in presence of CDKN1C mutations. The isolated form of the omphalocele accounts approximately for about the 14% of the total cases and its molecular etiology has never been fully elucidated. Methods: Given the tight relationship with BWS, we hypothesized that the isolated form of the omphalocele could belong to the heterogeneous spectrum of the BWS associated features, representing an endophenotype with a clear genetic connection. We therefore investigated genetic and epigenetic changes affecting BWS imprinted locus at 11p15.5 imprinted region, focusing in particular on the KCNQ1OT1:TSS DMR. Results: We studied 21 cases of isolated omphalocele detected during pregnancy or at birth and identified the following rare maternally inherited variants: i) the non-coding variant G > A at nucleotide 687 (NR_002728.3) at KCNQ1OT1:TSS-DMR, which alters the methylation pattern of the imprinted allele, in one patient; ii) the deletion c.624-629delGGCCCC at exon 1 of CDKN1C, with unknown clinical significance, in two unrelated cases. Conclusions: Taken together, these findings suggest that KCNQ1OT1:TSS-DMR could be a susceptibility locus for the isolated omphalocele. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is the presence of ESX1 mRNA in seminal fluid (SF) an indicator of residual spermatogenesis in men with non-obstructive azoospermic (NOA)? SUMMARY ANSWER ESX1 mRNA in SF is a suitable molecular marker for predicting the presence of residual spermatogenesis in testis. WHAT IS KNOWN ALREADY ESX1 is an X-linked homeobox gene whose expression in testis is restricted to germ cells. We previously reported, in the testicular biopsies from azoospermic men, a positive correlation between the presence of ESX1 mRNA and residual spermatogenesis. STUDY DESIGN, SIZE, DURATION We investigated ESX1 mRNA expression in 70 testicular fragments (TF) and 56 (SF) of 70 NOA men. As controls, we analyzed 8 TF from men with obstructive azoospermic (OA) and 9 SF from normozoospermic men. For all patients we considered the histological classification of testis biopsies and the recovery of spermatozoa by surgical procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS Relative ESX1 mRNA expression was evaluated by quantitative RT–PCR using the ΔΔCt method. The results were compared with the recovery of spermatozoa at surgery. MAIN RESULTS AND THE ROLE OF CHANCE In TF from NOA patients we found that: (i) ESX1 mRNA level was significantly decreased as the severity of spermatogenic defects increased (P < 0.0001, one-way analysis of variance); (ii) the presence of ESX1 mRNA can predict the success of sperm retrieval (sensitivity: 80%). In SF from NOA patients we found that: (i) ESX1 mRNA was present in 78.5% of NOA men; (ii) the presence of ESX1 mRNA could predict the success of sperm retrieval (sensitivity: 84%). LIMITATIONS, REASONS FOR CAUTION Spermatozoa were recovered at surgery in 5 out of 12 patients whose SF was negative for ESX1 mRNA expression. We think that discrepancies between molecular and clinical results could be reduced by analyzing more than one ejaculate from each man. WIDER IMPLICATIONS OF THE FINDINGS The data confirm that the ESX1 transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis. The implication of these results is that some patients ‘with azoospermia’, although having a severe impairment of spermatogenesis, could still maintain residual foci of spermatogenesis in limited areas of the testes, not always recovered by surgery. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico: Ricerca Corrente [grant number RC2014/519-02] to M.M. and from ASM onlus 2010–2011 to M.M. The authors declare that they have no conflict of interest. [ABSTRACT FROM AUTHOR]

Tuberous sclerosis complex ( TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis ( LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle ( ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/ TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/ TSC cells is epidermal growth factor ( EGF)-dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/ TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/m TOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/ TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/ TSC cells bear characteristics of stemness and secrete high amount of interleukin ( IL)-6 and IL-8. Anti- EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/ TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability. [ABSTRACT FROM AUTHOR]

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors. [ABSTRACT FROM AUTHOR]

Equivalent levels of X-linked gene products between males and females are reached by means of X chromosome inactivation (XCI). In the human and murine embryonic tissues, both the paternally and maternally derived X chromosomes (XP and XM) may be inactivated. In murine extra-embryonic tissues, XP is imprinted and always silenced; humans, unlike mice, can inactivate the XM in extra-embryonic lineages without an adverse outcome. This difference is probably due to the presence of imprinted placental genes on the murine X chromosome, but not on the human homologue, essential for placental development and function. An example is the paternally imprinted Esx1 gene; mice with a null maternally derived Esx1 allele show intrauterine growth restriction (IUGR) because of placental insufficiency. We investigated the imprinting status of the human orthologous Esx1 gene (ESX1L) in placental samples of four normal full-term and 13 IUGR female fetuses, in which we determined the XCI pattern. Our findings demonstrated that IUGR as well as normal placentas display XCI heterogeneity, thus indicating that the IUGR phenotype is not correlated with a preferential pattern of XCI in placentas. Moreover, ESX1L is equally expressed in IUGR and normal placentas, and shows the same methylation pattern in the presence of both random and skewed XCI. These findings provide evidence that ESX1L is not imprinted in human third-trimester placentas and there is no parent-of-origin effect of chromosome X associated with placental insufficiency.European Journal of Human Genetics (2004) 12, 272-278. doi:10.1038/sj.ejhg.5201121 Published online 3 December 2003 [ABSTRACT FROM AUTHOR]

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor β, RARβ, gene expression. Search of the causes affecting RARβ gene activity has been oriented at identifying possible differences either at the level of one of the RARβ promoters, RARβ2, or at regulatory factors. We hypothesized that loss of RARβ2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARβ2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARβ2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARβ2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR), led to demethylation within RARβ2 and expression of RARβ indicating that DNA methylation is at least one factor, contributing to RARβ inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARβ2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARβ2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARβ transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARβ activity, and help to overcome the hurdle of RA-resistance in breast cancer. Oncogene (2000) 19, 1556–1563. [ABSTRACT FROM AUTHOR]

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results. [ABSTRACT FROM AUTHOR]

Background: Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.Methods: To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.Results: We found severely (≥90:10) or moderately (≥75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations.Conclusions: Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers. [ABSTRACT FROM AUTHOR]

Understanding the relationship between genes and diet may improve cancer prevention and treatment. Genes and genomes seem able to adapt to dietary cues by epigenetic changes via postsynthetic modifications of either the DNA itself or the histone proteins that are intimately associated with DNA. We are beginning to understand the relationship among diet, individual gene differences (polymorphisms), and epigenetic modulation of genes and genomes and the direct influence of diet on the epigenetic status of genes and genomes. Vitamin A and its natural bioactive derivative retinoic acid (RA) have received a lot of attention for their potential anticancer effects on epithelial cells. However, in vitro studies and clinical trials using RA as a differentiation agent have shown that cancer cells are refractory to RA. RA action is normally mediated by retinoid receptors (RARs and RXR). RARs are transcriptionally active in the presence of both RA and activator-coactivator complexes with histone acetyltransferase activity. In contrast, they are repressed in the absence of RA by corepressor complexes with histone deacetylase activity. We found that RAR-beta is methylated in breast cancer cell lines and tumors (1) and that breast cancer patients with RAR-beta-methylated tumors do not respond to RA differentiation therapy (2). Recently, we found that RAR-beta methylation is present in normal epithelial cells adjacent to tumor cells in breast tissue, suggesting that epigenetic modifications leading to epigenetic RA resistance are likely an early event in the process of breast tumorigenesis. We hypothesized that factors leading to RAR-beta inactivity in human breast epithelial cells may predispose them to development of epigenetic RA resistance. When we forced RARbeta into transcriptional inactivity, we observed the appearance of epigenetic modifications at RAR-beta (DNA methylation, histone modifications) leading to RA resistance in breast epithelial cells. Our studies suggest that we may prevent the development of RA resistance in breast epithelial cells by preventing transcriptional inactivation of vitamin A signaling. Moreover, our studies show that we can reverse epigenetic changes leading to RA resistance and restore RA sensitivity in breast cancer cells. These studies may have a general validity for prevention and treatment of breast cancer as well as other epithelial cancers where we also observed the molecular signatures of epigenetic RA resistance. [ABSTRACT FROM AUTHOR]