Elissavet Hatzi

N- myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas.

Angiogenin Distribution in Human Term Placenta, and Expression by Cultured Trophoblastic Cells

Human angiogenin is a 14-kDa secreted protein with angiogenic and ribonucleolytic activities. Angiogenin is associated with tumour development but is also present in normal biological fluids and tissues. To further address the physiological role of angiogenin, we studied its expression in situ and in vitro, using the human term placenta as a model of physiological angiogenesis. Angiogenin was immunodetected by light and transmission electron microscopy, and its cellular distribution was established by double immunolabelling with cell markers including von Willebrand factor, platelet/endothelial cell adhesion molecule-1 (PECAM-1), CD34, Tie-2, vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Angiogenin immunoreactivity was detected in villous and extravillous trophoblasts, the trophoblast basement membrane, the endothelial basal lamina, foetal blood vessels, foetal and maternal red blood cells, and amnionic cells. Its expression was confirmed by in situ hybridisation with a digoxygenin-labelled cDNA probe and reverse transcriptase-polymerase chain reaction amplification. Villous cytotrophoblasts, isolated and differentiated in vitro into a functional syncytiotrophoblast, expressed and secreted angiogenin. Given its known biological activities in vitro and its observed pattern of expression, these data suggest that, in human placenta, angiogenin has a role not only in angiogenesis but also in vascular and tissue homeostasis, maternal immune tolerance of the foetus, and host defences.

The association of aromatase (CYP19) gene variants with sperm concentration and motility

The irreversible transformation of androgens into oestrogens is catalysed by cytochrome P450 aromatase. In the present study, we explored the contribution of the (TTTA)(n) polymorphism in the aromatase gene (CYP19) to sperm concentration and motility. Ninety normozoospermic and 60 oligospermic men were examined during infertility examinations. DNA was extracted from spermatozoa, and the CYP19 (TTTA)(n) polymorphism was genotyped by PCR. Genotype analysis revealed six CYP19 (TTTA)(n) alleles with 7-12 repeats. The allelic distribution of the CYP19 (TTTA)(n) polymorphism differed between normozoospermic and oligospermic men (P<0.01). Oligospermic men less frequently had long CYP19 alleles than did normozoospermic men (25 and 37.8%, respectively; P<0.02). The higher frequency of short CYP19 alleles in oligospermic men compared to normozoospermic men (43.3 and 28.3%, respectively; P<0.01) was primarily due to the distribution of the CYP19 (TTTA)(7) allele. The CYP19 (TTTA)(7) allele was associated with lower sperm concentration in normozoospermic men (P<0.01) and in the total study population (P<0.01); it was also associated with lower sperm motility in normozoospermic men (P<0.05) and in the total study population (P<0.01). In conclusion, the CYP19 (TTTA)(7) allele probably impairs aromatase activity, which in turn alters aromatase and oestrogen levels in the testis, leading to decreased sperm concentration and motility. These findings support the significance of cytochrome P450 aromatase in human spermatogenesis and consequently in semen quality.

Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization

PurposeSperm flow cytometry (SFC) was used to evaluate the association of sperm chromatin condensation and ploidy with fertilization, embryo development, pregnancy and abortion rates following IVF.MethodsConventional semen analysis was performed in one hundred fifty men, as well as SFC analysis, after acridine orange and propidium iodide staining, for the evaluation of sperm maturity and ploidy respectively. Conventional IVF was performed in all couples.ResultsCouples with low percentages of mature spermatozoa presented with lower fertilization rates (p < 0.005), lower rates of grade A embryos (p < 0.003) and lower pregnancy rates (p < 0.006), compared to couples with high percentages of mature spermatozoa. Couples with low total aneuploidy rates presented with higher fertilization rates (p < 0.007), higher rates of grade A embryos (p < 0.004) and higher pregnancy rates (p < 0.003), compared to couples with high total aneuploidy rates.ConclusionsSperm chromatin condensation and ploidy constitute critical parameters for the evaluation of semen samples before IVF and for the identification of cases in need of ICSI application.

MYCN Oncogene and Angiogenesis: Down-Regulation of Endothelial Growth Inhibitors in Human Neuroblastoma Cells

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis.

Association of serum and follicular fluid SHBG levels and SHBG (TAAAA)n polymorphism with follicle size in women undergoing ovarian stimulation

Objective. Sex hormone-binding globulin (SHBG) is the main transport protein of sex steroids. Recently, it has been found to be produced by granulosa lutein cells, suggesting a local role of SHBG in the ovary. The aim of this study was to investigate whether serum and follicular fluid SHBG levels and SHBG (TAAAA)n polymorphism are related to follicle size and pregnancy rate in women undergoing in vitro fertilisation. Methods. The study population consisted of 154 women with tubal and/or male-factor infertility undergoing IVF/ICSI and follicular fluid with oocytes from small (diameter ≤12 mm) and large (diameter ≥18 mm) follicles were studied. Genotyping of SHBG (TAAAA)n polymorphism was performed in peripheral blood samples. Serum and follicular fluids were used for hormones determination. Results. Women with short allele genotypes (with less than 8 TAAAA repeats) had higher number of small follicles compared to women with long allele genotypes (5.6 ± 3.9 vs. 3.5 ± 3.2 small follicles, p < 0.003). Follicular fluid SHBG levels correlated positively with serum SHBG levels (p < 0.001) and with the total number of follicles (p < 0.02). Furthermore, small follicles had higher follicular fluid SHBG concentration compared to large follicles (102.9 ± 35.0 nmol/l vs. 85.85 ± 34.88 nmol/l, p < 0.028). Conclusion. SHBG levels and the SHBG (TAAAA)n polymorphism are associated with follicle size.

Association of paraoxonase gene polymorphisms with sperm parameters.

Paraoxonase (PON) is a high-density lipoprotein-associated enzyme that prevents low-density lipoprotein oxidation. PON proteins, localized in the seminiferous tubules and in spermatozoa, have been implicated in the pathogenesis of male infertility. In the present study, we sought to explore the contribution of the PON gene variants to sperm parameters. One hundred twenty oligospermic and 170 normozoospermic men were examined during infertility investigation. DNA was extracted from spermatozoa, and the PON1(L/M) 55, PON1(Q/R) 192, and PON2(S/C) 311 polymorphisms were genotyped by polymerase chain reaction and digestion with restriction enzymes. The analysis revealed that oligospermic men presented PON1 55L/L, PON1 192Q/Q, and PON2 311S/S genotypes less frequently than normozoospermic men (P < .01, P < .01, and P < .001, respectively), whereas the PON1 55M, PON1 192R, and PON2 311C alleles were significantly increased in oligospermic men (P < .004, P < .008, and P < .008, respectively). The presence of PON1 55L allele was associated with higher sperm motility in oligospermic men (P < .001), in normozoospermic men (P < .01), and in the total study population (P < .01), and the PON1 192Q allele was associated with higher sperm motility in oligospermic men (P < .01), in normozoospermic men (P < .04) and in the total study population (P < .03). On the other hand, the PON2 311S was associated with higher sperm concentration in oligospermic men (P < .03), in normozoospermic men (P < .008), and in the total study population (P < .001). In our series, the PON1 55M and PON1 192R alleles were associated with decreased sperm motility whereas the PON2 311C allele was associated with decreased concentration, supporting the significance of PON genes in semen quality.

Using semen flow cytometry to evaluate association of ploidy status and chromatin condensation of spermatozoa with conventional semen parameters: Clinical application in intrauterine insemination

OBJECTIVE To evaluate the association between the ploidy status and the nuclear chromatin condensation of spermatozoa with conventional semen parameters by using semen flow cytometry (SFC). The prognostic value of SFC on the successful outcome of intrauterine insemination (IUI) was examined. DESIGN Prospective study. SETTING Patients referred to the IVF Unit of Ioannina University School of Medicine. PATIENT(S) Ninety-two men with sperm count between 15×10(6) and 40×10(6) spermatozoa/mL and one hundred normozoospermic men were analyzed. INTERVENTION(S) Conventional semen analysis and SFC analysis after acridine orange and propidium iodide staining. IUI performed in 92 couples. MAIN OUTCOME MEASURE(S) Evaluation of sperm maturity and ploidy. Correlation with conventional semen parameters. RESULT(S) An association of the spermatozoa ploidy status with sperm morphology and motility was revealed. The highest aneuploidy rates were observed when <15% of spermatozoa had normal morphology. An inverse relation was found between sperm morphology and maturity. The pregnancy rates were significantly lower when semen with <15% normal forms (9% vs. 25%), low percentage of mature spermatozoa, and increased total aneuploidy rate were used for IUI. CONCLUSION(S) Ploidy status and sperm maturity are critical parameters for evaluation of the fertilizing capacity of spermatozoa. SFC could be used to evaluate semen samples before IUI and potentially prognose the outcome.

The ovarian response to standard gonadotrophin stimulation depends on FSHR, SHBG and CYP19 gene synergism

PurposeFollicle stimulating hormone, sex hormone-binding globulin and cytochrome P450 aromatase play crucial roles in the regulation of mammalian reproduction. The synergistic effect of FSHR 307(T/A)/FSHR 680(N/S), SHBG(TAAAA)n and CYP19(TTTA)n genotypes on ovarian response to standard gonadotrophin stimulation of women undergoing medically assisted reproduction (IVF/ICSI) was explored.MethodsThe study population consisted of 300 women under IVF/ICSI treatment and 300 women with at least with at least one successful child birth as controls. The polymorphisms were genotyped while the follicular size, the follicle and oocyte numbers were recorded during oocyte retrieval.ResultsThe genotype analysis, excluding heterozygotes for each particular polymorphism, revealed eight combined homozygotic FSHR/SHBG/CYP19 genotypes. A gradual reduction in the number of follicles and oocytes from FSHR 307Thr/680Asn allele/long SHBG allele/long CYP19 allele homozygotes to FSHR 307Ala/680Ser allele/short SHBG allele/short CYP19 allele homozygotes was observed (20.36 ± 6.74 vs. 8.05 ± 2.47, p < 0.008 and 13 ± 4.63 vs. 6.1 ± 2.32, p < 0.02, respectively).ConclusionsFSHR/SHBG/CYP19 combined genotypes are associated with ovarian response to standard gonadotrophin stimulation of women undergoing medically assisted reproduction.

CYP19 gene variants affect the assisted reproduction outcome of women with polycystic ovary syndrome

Abstract Objective: Cytochrome P450 aromatase catalyzes the irreversible transformation of androgens into estrogens. The association of CYP19(TTTA)n polymorphism with the hormonal profile and the assisted reproduction outcome of women with polycystic ovary syndrome (PCOS) was explored. Methods: One hundred and thirty-two women with PCOS and 200 with male-factor infertility, as controls, participated in the current study. The CYP19(TTTA)n polymorphism was genotyped, while the hormonal profile was determined at the third day of the menstrual cycle. During oocyte retrieval, the follicular size, the follicle and oocyte numbers were recorded. Results: Genotype analysis revealed 6 CYP19(TTTA)n alleles with 7–12 repeats. In PCOS women, the CYP19(TTTA)7 allele presence was associated with lower serum E2 levels at the third day of the menstrual cycle (p < 0.009), lower large follicle (p < 0.02) and total oocyte numbers (p = 0.006), but with significantly higher pregnancy rates after assisted reproduction (p < 0.004). Conclusions: Potential associations of the CYP19(TTTA)7 allele with ovarian response to standard gonadotrophin stimulation and with assisted reproduction outcome were found in PCOS women, probably due to androgen/estrogen ratio alterations.