David Agudo

Nup88 mRNA overexpression is associated with high aggressiveness of breast cancer

The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Immunohistochemical studies have shown that this overexpression is linked to higher aggressiveness of colorectal carcinoma and to enhanced metastatic potential of melanoma cells. However, the antibodies so far developed against Nup88 have the drawback of recognizing a number of other, up to now unspecified antigens besides Nup88. For this reason, we devised the present study on Nup88 expression at the mRNA level. RNA was extracted from fresh tumor tissue corresponding to 122 breast cancer patients. Nup88 mRNA expression was measured by means of differential RT‐PCR, standardizing against a constitutive internal control gene (β‐actin). The results were dichotomized into “high” and “low” expression levels, using the median value as cut‐off. High Nup88 mRNA expression levels correlated significantly with ductal and tubular histology (p = 0.012), histologic and nuclear grade 3 of tumors (p < 0.001), absence of hormone receptor expression (p < 0.001), expression of the c‐erb‐B2 oncogene (p < 0.001), expression of mutant p53 protein (p < 0.001), high proliferation (defined by Ki67 labeling index >20%, p < 0.001), DNA aneuploidy (p < 0.001) as well as the most important ominous clinical prognostic factor, axillary node invasion (p < 0.001). We also found an inverse correlation (p < 0.001) with expression of the H‐MAM (mammaglobin) gene, a marker of low biologic and clinical aggressiveness of breast cancer. All of these factors, without exception, define a highly aggressive tumor phenotype. These findings appear to be specific to Nup88 and not to nuclear pore proteins in general. Indeed, analysis of Nup107 (which is a limiting component of the nuclear pore complex) under the same conditions in the same tumors did not yield comparable results.

Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X‐chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT‐PCR. Using the same method, we also studied the expression of the apoptosis‐related genes Bcl‐2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA‐ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c‐erb‐B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X‐chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P = 0.039; RBM3, P < 0.001; RBM10 large variant, P < 0.001; RBM10 small variant, P < 0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P = 0.004; small variant, P = 0.003). We also found an association of borderline significance (P = 0.05) between the expression of RBM3, the large variant of RBM10 and wild‐type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67 ≥ 20%; P = 0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r = 0.26, P = 0.006). From these results, it seems that the X‐chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer. J. Cell. Biochem. 97: 1275–1282, 2006.

FSH receptor in vitro modulation by testosterone and hCG in human luteinized granulosa cells

OBJECTIVE To investigate the effect of testosterone and hCG on FSH receptor (FSHR) protein and mRNA expression in human granulosa cells (GC) in vitro. STUDY DESIGN Experimental in vitro cell culture obtained from healthy women undergoing IVF/ICSI due to male factor infertility. Human follicular fluid samples were obtained and after cumulus-oocyte complexes were identified, fluids were pipetted onto Ficoll gradients and centrifuged for 15 min at 400 × g at room temperature. Cells at the interface were removed and plated in 24-well plates for 3 days in M-199 with 10% FBS. Cells were treated with different concentrations of testosterone and hCG. After purification, cells were labeled with specific antibodies and the protein expression of the FSHR was evaluated by flow cytometry in the GC population. Also, total RNA was extracted from confluent GC and the FSHR gene expression was evaluated by RT-PCR. RESULTS FSHR expression was modulated by treating GC in vitro at different testosterone/hCG concentrations. When compared with untreated GC, we observed a significant effect of testosterone and hCG on the expression of the FSHR at the protein level. Time course experiments confirmed that the gene expression of the FSHR peaked at 12-24h when testosterone or hCG was used as a stimulus. CONCLUSIONS Both testosterone and hCG are able to positively modulate FSHR expression at gene and protein level in human GC in vitro.

Type of gonadotropin during controlled ovarian stimulation affects the endocrine profile in follicular fluid and apoptosis rate in cumulus cells.

OBJECTIVE To determine whether the type of gonadotropin affects the secretion of oocyte-specific factors, the endocrine pattern in follicular fluid, and the apoptosis rate in cumulus cells. STUDY DESIGN Prospective and observational study into an university-affiliated private in vitro fertilization setting. Ninety women included in our oocyte donation program were stimulated with human menopausal gonadotropin (hMG), recombinant follicle-stimulating hormone (FSH) or urinary FSH. Main outcome measures were growth-differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) expression, hormonal profile and apoptosis rate. RESULTS No statistically significant differences were observed for GDF-9 and BMP-15 among the three treatment groups. Estradiol concentrations in follicular fluid were significantly higher in women treated with hMG compared with recombinant FSH or urinary FSH. Testosterone levels were also higher in the group treated with hMG. A statistically significant association was found between the degree of apoptosis in cumulus cells and the type of gonadotropin. CONCLUSIONS The type of gonadotropin used during controlled ovarian stimulation significantly affects endocrine profiles in follicular fluid and the apoptosis rate in cumulus cells. However, there were no significant differences in the levels of oocyte-secreted factors between treatments.

Type of gonadotropin used during controlled ovarian stimulation induces differential gene expression in human cumulus cells: A randomized study

BACKGROUND The cumulus-oocyte complex plays a central role in the regulation of folliculogenesis where it is important for the maturation, reprogramming, and fertilization of oocytes. Consequently, cumulus cell gene expression profiling is being explored as a promising method for assessing oocyte competence in the near future. Through DNA microarray technology, we analyzed the potential differences in the gene expression profiles of cumulus cells from preovulatory follicles after controlled ovarian stimulation using different types of gonadotropins. METHODS A prospective, randomized study was performed among 90 women participating in an oocyte donation program. Subjects were assigned to receive recombinant follicle-stimulating hormone (FSH), urinary FSH, or human menopausal gonadotropin (hMG). The gene expression profile in cumulus cells was analyzed according the type of gonadotropin received during ovarian stimulation. Furthermore, we also performed a gene ontology analysis to provide structural knowledge. RESULTS Hierarchical clustering, principal component analysis, and gene enrichment analysis revealed greater differences between the urinary FSH and hMG groups compared to the rest of the pair-wise comparisons; recombinant FSH vs hMG and urinary FSH vs recombinant FSH. CONCLUSIONS Data suggest that controlled ovarian stimulation induces specific gene expression profiles in human cumulus cells depending on the type of gonadotropin used. TRIAL REGISTRATION Registered at clinicaltrials.gov; identifier NCT022437032.

Should we forget about embryos till day 5

Purpose of review To find the way of having more and better blastocyst is essential. How to culture embryos up to blastocyst stage remains critical. Recent findings Several studies show how a blastocyst score can predict the implantation potential. If that score is enough to choose the best blastocyst, as culture conditions would not be affected in these days, we would not need to check early cleavage embryos, even it could be better for the embryo development. Summary The item that should be discussed is if it is better to evaluate or not embryos at early cleavage stages. If we do not check embryos on days 2 and 3, we should change our way to work and how to culture those embryos. First step would be to perform all embryo transfers on day 5 or 6. If we let embryos grow to blastocyst without any morphology evaluation, we should adapt several steps in our laboratory, for example we should move to a single-step culture medium or we should not do assisted hatching on day 3 embryos.