Combined treatment with myo-inositol, alpha-lipoic acid, folic acid and vitamins significantly improves sperm parameters of sub-fertile men: a multi-centric study.

OBJECTIVE: Reduction in motility and number of spermatozoa and change in their morphology are some of the most relevant causes of male infertility. Production of reactive oxygen species may affect motility, morphology and DNA stability of spermatozoa. This study aimed at evaluating the effect of combined treatment with myo-inositol, alpha-lipoic acid, folic acid, betaine and vitamins (namely, Sinopol®) on semen parameters of sub-fertile men. PATIENTS AND METHODS: We recruited 143 sub-fertile men, 26-53 years aged, no-smokers, without any testicular pathologies, with a normal endocrinological/metabolic profile, and no concomitant consumption of drugs. Out of them, 25 patients did not meet study inclusion criteria mainly due to the history of genital diseases that came to light after Sinopol® prescription. Among the 118 men that fulfilled inclusion criteria, 10 (8.4%) patients were lost at follow-up and in 8 (6.8%) cases the partner got pregnant spontaneously. Thus, 100 patients completed the study and semen analysis was performed before and after 90 days of treatment. RESULTS: Semen quality improved after 90 days of treatments, with a statistically significant increase of sperm concentration (p=0.0009), of number of spermatozoa (p=0.0017), of progressive motility (p=0.0047), of total motile sperm count (p=0.0010), and of normal sperm morphology (p<0.0001). CONCLUSIONS: For the first time we reported that a combination of nutraceuticals composed of myo-inositol, alpha-lipoic acid, folic acid, betaine and vitamins improves sperm parameters in sub-fertile men. We are aware that to clarify the clinical relevance of the data studies with larger sample sizes and longer durations are needed, as well as evaluation of myo-inositol and alpha-lipoic acid co-treatment effectiveness in improving the chances to obtain a pregnancy spontaneously or following assisted reproduction.

Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome.

In assisted reproduction technology, embryo competence is routinely evaluated on morphological criteria. Over the last decade, efforts in improving non-invasive embryo assessment have looked into the secretome of embryos. Human embryos release genomic DNA (gDNA) and mitochondrial DNA (mtDNA) into the culture medium, and the mtDNA/gDNA ratio is significantly correlated with embryo fragmentation. Here, we investigate whether mtDNA/gDNA ratio in embryo spent medium is correlated with blastulation potential and implantation. The mtDNA/gDNA ratio was assessed in 699 Day 3 culture media by quantitative polymerase chain reaction (qPCR) to investigate its correlation with embryo morphology, blastocyst development and implantation. A logistic regression model evaluated whether mtDNA/gDNA ratio in the secretome may improve the prediction of blastulation. We found that embryos that successfully developed into blastocysts exhibited a significantly higher mtDNA/gDNA ratio in the culture medium compared with those that arrest (P = 0.0251), and mtDNA/gDNA, combined with morphological grading, has the potential to predict blastulation better than morphology alone (P = 0.02). Moreover, mtDNA/gDNA ratio was higher in the media from good-quality embryos that reached the full blastocyst stage on Day 5 compared with those that developed more slowly (P < 0.0001). With respect to blastocyst morphology, higher trophectoderm quality was associated with a higher mtDNA/gDNA ratio in the culture medium. Finally, a high mtDNA/gDNA ratio in spent medium was associated with successful implantation outcome (P = 0.0452) of good-quality embryos. In summary, the mtDNA/gDNA ratio in the Day 3 embryo secretome, in combination with morphological grading, may be a novel, non-invasive, early biomarker to improve identification of viable embryos with high developmental potential.

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

STUDY QUESTION: Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? SUMMARY ANSWER: The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. WHAT IS KNOWN ALREADY: Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. STUDY DESIGN, SIZE, DURATION: Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. MAIN RESULTS AND THE ROLE OF CHANCE: Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. LIMITATIONS, REASONS FOR CAUTION: It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. WIDER IMPLICATIONS OF THE FINDINGS: The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. STUDY FUNDING/COMPETING INTEREST: This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT01397136.

Genomic aberrations in normal appearing mucosa fields distal from oral potentially malignant lesions.

OBJECTIVES: Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the "field carcinogenesis" model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy. METHODS: Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs). RESULTS: The frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31-q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31-q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01). CONCLUSIONS: ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31-q13.33 may contribute to DNA aneuploidization.

DNA-functionalized solid state nanopore for biosensing.

The possible use of nanopores for single DNA molecules biosensing has been demonstrated, but much remains to do in order to develop advanced engineered devices with enhanced stability, and controlled geometry and surface properties. Here we present morphological and electrical characterization of solid state silicon nitride nanopores fabricated by focused ion beam direct milling and chemically functionalized by probe oligonucleotides, with the final aim of developing a versatile tool for biosensing and gene expression profiling.

Electrical characterization of DNA-functionalized solid state nanopores for bio-sensing.

We present data concerning the electrical properties of a class of biosensor devices based on bio-functionalized solid state nanopores able to detect different kinds of interactions between probe molecules, chemically attached to the pore surface, and target molecules present in solution and electrophoretically drawn through the nanometric channel. The great potentiality of this approach resides in the fact that the functionalization of a quite large pore (up to 50-60 nm) allows a sufficient diameter reduction for the attainment of a single molecule sensing dimension and selective activation, without the need for further material deposition, such as metal or oxides, or localized surface modification. The results indicate that it will be possible, in the near future, to conceive and design devices for parallel analysis of biological samples made of arrays of nanopores differently functionalized, fabricated by standard lithographic techniques, with important applications in the field of molecular diagnosis.

Genome and transcriptome analysis of neuroblastoma advanced diagnosis from innovative therapies.

Neuroblastoma is an extracranial solid tumor which occurs in infants and young children and accounts for 8% of pediatric cancers. It origins from neural crest cells of the sympathetic nervous system. Disease-free survival ranges from 95% for localized tumors to 30% for metastatic disease in children over 1 year of age and patients' outcome depends on dissemination and tissue histology. Despite the most recent therapies, the overall survival for high risk patients is still low and the outcome is invariably fatal. Improvement of neuroblastoma treatment is one of the highest priorities in pediatric oncology and a major challenge to clinicians and researchers. Understanding the biology and genetics of pediatric malignancies will be the key to identify molecular targets for innovative treatments as well as to individual management of disease. The success of human genome project and recent advances in technology have provided new tools to investigate cancer cells and to discover new tumor-associated genes. High-throughput efforts include array-based comparative genomic hybridization, single-nucleotide polymorphism arrays and expression microarrays. Here we present an overview on the most recent advances in wide-genome analysis of neuroblastoma. We also focus on the potential clinical application of genome and transcriptome information to the diagnosis, prognosis and neuroblastoma therapy.

Biological and clinical role of p73 in neuroblastoma.

The p73 gene is a p53 homologue localized at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. p73 was originally considered an oncosuppressor gene. However, it was soon realized that its mode of action did not resemble that of a classic anti-oncogene. The recent discovery of N-terminal truncated isoforms, with oncogenic properties, showed that p73 has a 'two in one' structure. Indeed, the full-length variants are strong inducers of apoptosis while the truncated isoforms inhibit the pro-apoptotic activity of p53 and of the full-length p73. This review summarizes some aspects of p73 biology with particular reference to its possible role in neuroblastoma.

Quantitative determination of sst2 gene expression in neuroblastoma tumor predicts patient outcome.

Neuroblastoma (NB) is the most common pediatric neuroendocrine tumor, and it is characterized by a quite variable clinical course. We previously found a great variability in the expression of somatostatin receptor type 2 (sst2) in several human NB cell lines and primary tumors. In this report we investigated whether expression of sst2 is somehow related to clinical outcome. We performed a retrospective study on 54 patients with a maximum follow-up of 100 months. The concentration of specific messenger ribonucleic acid (mRNA) for sst2 was measured by competitive RT-PCR and validated, in a small subset of samples, by quantitative imaging of gene (in situ hybridization) and protein (immunohistochemistry) expression. We found that sst2 mRNA was variably expressed in all NB tumors (range, 2.5 x 10(5) to 8 x 10(9) molecules/microg RNA) with a relevant reduction in the more advanced stage (P < 0.01). Analysis of Kaplan-Meier curves indicated that sst2 expression is positively related to the overall (P < 0.0001) and event-free (P < 0.0001) survival. Expression of sst2 was negatively related to tumor stage (P < 0.02) and MYCN amplification (P < 0.001), a poor prognostic factor. However, the prognostic information derived from sst2 is apparently independent from MYCN amplification, as assessed by stratifying sst2 values according to MYCN. In addition, the expression of sst2 was the only significant prognostic factor (P < 0.02) when it was included in a multivariate model containing other well known prognostic factors such as age, stage, and MYCN amplification. Hence, we propose that sst2 expression represents a new prognostic marker for NB. The main clinical value of a quantitative measure of sst2 lies in its ability to detect patients at low risk, independently from other prognostic factor, including MYCN amplification.

Stage-independent expression and genetic analysis of tp73 in neuroblastoma.

The tp73 gene, a tp53 homologue, has been sub-regionally mapped at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. Due to its chromosomal localization and to the mono-allelic expression observed in some neuroblastoma cell lines, it was proposed that tp73 might be involved in the pathogenesis of neuroblastoma. Functional assays have demonstrated that tp73 can inhibit cell proliferation and induce apoptosis. The role of this gene in tumorigenesis, however, is still unclear. We analyzed tp73 expression in 95 sporadic neuroblastoma samples by RT-PCR and we detected the tp73 transcript in 46 cases (48.4%), without significant correlation with age, clinical stage or 3-year overall survival. A genetic polymorphism in the 2nd exon of tp73 was utilized to identify the transcribed allele in tumor-cell samples. Expression from only one of the tp73 alleles was found in 13 out of 16 heterozygous tumors, while in 3 samples both alleles were present. Genotype analysis of 73 patients and 150 controls showed a significant deviation (p = 0.0308) from the Hardy-Weinberg equilibrium for a tp73 allele only among neuroblastoma patients. The absence of correlation between tp73 expression and clinical stage, age and survival suggests that this gene does not play an essential function in the clinical course of the disease. However, the distribution of genomic tp73 alleles in patients indicates that a role of this gene in the development of neuroblastoma cannot be completely ruled out. Int. J. Cancer (Pred. Oncol.) 84:365-369, 1999.

Detection of DNA polymorphisms and point mutations of high-affinity nerve growth factor receptor (TrkA) in human neuroblastoma.

We have studied nerve growth factor (NGF) receptors in 63 neuroblastoma tissues. We found DNA polymorphisms of TrkA consisting in C-->T transitions in tyrosine kinase domain at 6773, 7232 and 7301 nucleotides. Both C/T alleles were detected in tumors belonging to patients at stages 1, 2, 3 and 4 but not in 4S that expresses only the allele with C-->T. Furthermore, we detected a GT transversion resulting in a Gly-->Val substitution in a stage 4 and a stage 4S samples. We report the first evidence of TrkA polymorphism and point mutations in neuroblastoma.

[The identification of minimal residual disease in the bone marrow and peripheral blood in neuroblastoma. The prognostic and therapeutic implications]. / Identificazione della malattia residua minima nel midollo e nel sangue periferico nel neuroblastoma. Implicazioni prognostiche e terapeutiche.

A highly sensitive and specific methodology to detect neuroblastoma cells in the bone marrow and peripheral blood of children with neuroblastoma is of critical importance for proper staging and treatment of these patients. In addition, patients with bone marrow infiltration at diagnosis need to undergo regular investigation to measure the effectiveness of chemotherapy (so called "in vivo" purging). Finally, the evaluation of autologous stem cells taken from bone marrow or peripheral blood is necessary to rule out or minimise the possibility of reinfusing tumor cells to the patient following myeloablative therapy. The authors provide a "state of the art" data on this complicated issue and give their preliminary results of their own experience, mainly concerning the immunocytological methods.

MYCN oncogene amplification in neuroblastoma is associated with worse prognosis, except in stage 4s: the Italian experience with 295 children.

PURPOSE: To evaluate the prognostic role of MYCN oncogene amplification in children with neuroblastoma. PATIENTS AND METHODS: Of 694 children (age, 0 to 15 years) with previously untreated neuroblastoma, 295 (42%) were evaluated at diagnosis for MYCN gene amplification. RESULTS: Clinical characteristics and survival results of 295 patients studied and 399 not studied for MYCN were comparable. In 48 of 295 patients studied for MYCN (16%), the gene was amplified (> or = three gene copies). Amplification was more frequent in children older than 1 year, with abdominal tumor (18% v 7%), advanced disease, normal vanillylmandelic (VMA) urinary excretion, and high lactate dehydrogenase (LDH), ferritin, and neuron-specific enolase (NSE) serum levels. In patients studied for MYCN, the 5-year overall survival (OS) rate was higher for children aged less than 1 year (90% v 44%), with extraabdominal tumor, stage 1 or 2 versus 3 versus 4, and normal NSE, LDH, and ferritin serum levels. Patients with amplified MYCN had a worse OS (odds ratio [OR], 3.38; confidence interval [CI], 2.22 to 5.16). This association held after adjustment for other characteristics. The impact of MYCN amplification was greater in patients with favorable characteristics, in particular age (OR, 10.28 for infants; 2.08 for older children) and stage (OR, 35.3 for stage 1 to 2; 8.41 for stage 3; 1.76 for stage 4). However, of 29 children with stage 4s, all three with amplified MYCN survive. In a multivariate analysis, the prognostic role of MYCN amplification, age, and stage was confirmed, but the size of the effect of MYCN was dependent on age and stage. CONCLUSION: MYCN amplification is associated with a worse prognosis in children with neuroblastoma at all ages and stages except 4s. This association is most pronounced in children with otherwise favorable prognostic indicators, and in these children should be considered as an indication for more intensive intervention.

Restriction fragment length polymorphism analysis reveals different allele frequency and a linkage disequilibrium at locus D1S94 in neuroblastoma patients.

Deletion of chromosome 1p and MYCN amplification have been reported as frequent abnormalities in human neuroblastoma. We studied loss of heterozygosity (LOH) in 50 (48 informative) Italian neuroblastoma patients by restriction fragment length polymorphisms (RFLPs) analysis using anonymous and hypervariable region (HVR) sequences. Twelve cases (25%) showed LOH at one or more loci. Locus D1S94 was the most frequently involved in LOH events (8/12) of deleted cases (66.6%). MYCN amplification was observed in 20% of patients which showed a significantly lower event-free survival probability (EFSp) (P = 0.004). We also studied the allelic distribution in the constitutional DNA of neuroblastoma patients (n = 44) and a matched group of healthy Italian subjects (n = 79) for loci D1S112 and D1S94. A significantly (P = 0.01) different allele frequency was detected for the two groups at locus D1S94, but not at D1S112. Moreover, the neuroblastoma population did not confirm the Hardy-Weinberg expectations at the former locus. This observation suggests the existence of an allelotype associated with neuroblastoma susceptibility.

Loss of heterozygosity for chromosome 1p in familial neuroblastoma.

Loss of heterozygosity (LOH) and deletion of chromosome 1p are very often found in sporadic neuroblastoma. Nevertheless, very few data are available concerning 1p LOH in familial neuroblastoma. Families with recurrent neuroblastoma are rare and analysis of chromosome 1p in these families might give useful information for identifying the putative neuroblastoma suppressor gene. We used combined cytogenetic and molecular techniques to study 1p LOH in two neuroblastoma families. Family M has 2 out of 3 children with neuroblastoma and family C has 2 children, 1 of whom has neuroblastoma and type 1 neurofibromatosis (NF1). All patients of both families showed tumour cells with chromosome 1p deletion (1pdel), but only the patient from family C also had MYCN gene amplification. In all cases the deleted chromosome 1 was of maternal origin.

Expression of MAGE-1, MAGE-3 and MART-1 genes in neuroblastoma.

The human MAGE-1, MAGE-3 and MART-1 genes code for antigens that are specifically recognized by cytolytic T lymphocytes in a MHC-restricted manner. The MAGE-1 and MAGE-3 genes are expressed in tumors of different histotypes but not in normal adult tissues (with the exception of testis), while the MART-1 gene appears to be selectively expressed in melanoma. MAGE-1, MAGE-3 and MART-1 antigens may therefore constitute useful targets for specific anti-tumor immunization of cancer patients. Here we have investigated the expression of MAGE-1, MAGE-3 and MART-1 in 11 neuroblastoma (NB) cell lines and 73 NB tumor masses. MAGE-1 and MAGE-3 transcripts were detected simultaneously in 36% of the cell lines and in 16% of tumor samples. The MAGE-1 gene was never expressed alone except in one tumor. In contrast, MAGE-3 mRNA was found in approximately 40% of the NB tumor samples in the absence of MAGE-1 mRNA. No expression of the MART-1 gene was observed in any cell line or tumor sample. No correlation was found between MAGE gene expression and clinical stage, event-free survival and presence or absence of N-myc amplification.

Gene expression and protein localisation of calcyclin, a calcium-binding protein of the S-100 family in fresh neuroblastomas.

Calcyclin gene, a Ca(2+)-binding protein with homology to S-100, has been found to be expressed at different levels in leukaemic cells and in other tumour cells. We recently reported the expression of the gene in human neuroblastoma (NB) cell lines, and suggested a possible role of calcyclin in cell differentiation. To extend our findings, we investigated the expression of the gene in NB cells induced to differentiate by retinoic acid (RA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Time-course experiments employing LA-N-5 cells showed that calcyclin mRNA appeared 2 h after RA treatment, long before the cells were blocked in the G1 cell-cycle phase and before the neurite-like structures outgrew from the cell bodies. This suggests the involvement of the gene in the early phase of cell differentiation. Furthermore, we investigated mRNA expression in a series of fresh neuroblastomas. NB tumours showed a heterogeneous pattern of calcyclin expression, although calcyclin seemed to be expressed more frequently in cases with a favourable Shimada histology. We also studied the expression of the protein in formalin fixed and paraffin embedded tissues, by using a specific anticalcyclin antibody. The protein was detected in stromal cells which characterise a more mature histological type, and in nerve sheaths, whereas neuroblasts were negative. The tissue that expressed calcyclin protein showed a Schwann-like differentiation and, unlike S-100 protein, calcyclin was expressed in the perineurium.

[Oncogenes and suppressor genes in the genesis and progression of solid tumors in children]. / Oncogeni e geni soppressori nella genesi e nella progressione dei tumori solidi del bambino.

Molecular and genetic analyses of tumor cell show that cellular oncogenes and suppressor genes are involved in neoplastic transformation. In pediatric tumors oncogenes as N-myc play an important role in the tumor progression. In retinoblastoma, neuroblastoma, Wilms' tumor, and rhabdomyosarcoma loss of heterozygosity for specific chromosome loci has been suggested to be a critical step in cancer development. Oncogene abnormalities can also be useful as a molecular tumor factor to foresee the prognosis of the disease. The present article is a review on the role of the oncogenes and suppressor genes in pediatric solid tumors.