A freeze-all strategy does not increase live birth rates in women of advanced reproductive age.

RESEARCH QUESTION: Does a freeze-all strategy improve live birth rates in women of different age groups? DESIGN: Retrospective cohort study of 1882 first embryo transfer cycles, performed between January 2013 and December 2015. Reproductive outcomes between fresh (FRESH) or frozen (FROZEN) embryo transfers were compared in patients stratified by age: < 35, between 35 and 38, or > 38 years. Student's t test for independent samples and χ2 analyses were used as needed. A multivariable logistic regression analysis was performed adjusting for age, triggering drug, number of retrieved oocytes, number of transferred embryos, and percentage of top-quality embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Live birth rates (LBR) were significantly higher for FROZEN in the < 35 years group (43.7% vs 24%; p < 0.001). In both the 35-38 and > 38 years groups, LBR for FROZEN vs FRESH were not statistically different (30.9% in the FROZEN group vs 29.3% in the FRESH group, p = 0.70, and 19.8% in the FROZEN group vs 12.7% in the FRESH group, p = 0.07, respectively). The multivariate analysis found a significantly positive effect of performing FROZEN on LBR in the younger group (OR 2.46, 95% CI 1.31-4.62; p = 0.005) but had no impact in women between 35 and 38 years (OR 1.01, 95% CI 0.55-1.83; p = 0.98) or older (OR 0.96, 95% CI 0.43-2.13; p = 0.92). CONCLUSIONS: Performing a freeze-all strategy seems to result in better reproductive outcomes when compared with a fresh ET in women under 35 years, with no significant impact on older women.

Sperm telomere length in motile sperm selection techniques: A qFISH approach.

Several studies have associated telomere shortening with alterations in reproductive function. The objective of the present study was to determine telomere length (TL) in spermatozoa selected by either density-gradient centrifugation (DGC) or swim-up. The analysis of TL was performed using quantitative fluorescent in situ hybridisation (qFISH) using PNA probes in combination with a chromatin decompaction protocol in sperm cells. Results of TL were 24.64 ± 5.00 Kb and 24.95 ± 4.60 Kb before and after DGC, respectively, and 19.59 ± 8.02 Kb and 20.22 ± 5.18 Kb before and after swim-up respectively. Sperm selected by DGC or swim-up did not show any significant differences in TL as compared to nonselected sperm (p > .05). Negative correlations between TL and sperm motility (r = -.308; p = .049) and concentration (r = -.353; p = .028) were found. Furthermore, exposure of sperm to increasing concentrations of hydrogen peroxide during incubation resulted in a reduction in TL. These data indicate that oxidative stress may be one of the main factors involved in the reduction of TL in sperm. Preliminary clinical results from patients included in this study indicate that TL was shorter in spermatozoa from couples who never achieved a pregnancy compared to couples who did achieve at least one natural pregnancy (p < .05); however, the clinical utility of this biomarker still needs to be confirmed in further studies.

There is no evidence that the time from egg retrieval to embryo transfer affects live birth rates in a freeze-all strategy.

STUDY QUESTION: Does the time from ovum pick-up (OPU) to frozen embryo transfer (FET) affect reproductive outcomes in a freeze-all strategy? SUMMARY ANSWER: Our study did not detect statistically significant differences between first and subsequent cycles, clinically relevant differences are not ruled out and further and larger studies are required. WHAT IS KNOWN ALREADY: Following controlled ovarian hyperstimulation (COH) delaying FET until the endometrium has returned to an optimal pre-stimulation state may have a significant emotional impact on patients, which adds to the stress and anxiety accompanying a standard IVF cycle. Currently there is no agreement on the best time to perform a FET after a freeze-all cycle in order to maximize reproductive outcomes for the patient. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of 512 freeze-all cycles, performed between January 2012 and December 2014. COH was performed by either a GnRH antagonist (n = 397) or a long GnRH agonist protocol (n = 115). Ovulation was triggered using either a GnRH agonist (n = 258) or hCG (n = 254). Endometrial preparation was performed in an artificial cycle by either oral (n = 238) or transdermal (n = 274) oestrogen. Differences were considered significant if P < 0.05. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive outcomes between FETs which took place either within the first menstrual cycle following OPU (Cycle 1; n = 263) or afterwards (Cycle ≥2; n = 249) were compared. Student's t-test for independent samples, Mann-Whitney U-test and Chi-square analysis were used where appropriate. A multivariable logistic regression analysis was performed adjusting for maternal age, drug used for ovulation trigger, number of retrieved oocytes, number of embryos obtained, day of embryonic development at transfer, number of embryos transferred and type of endometrial preparation. Differences were considered significant if P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Live birth rate (LBR) was significantly higher in FET performed during Cycle 1 vs Cycle ≥2 (37.6% vs 27.3%, respectively; P = 0.01) before adjusting for confounding factors. We found no difference for biochemical pregnancy (49.8% vs 43.8%; P = 0.17), clinical pregnancy (44.1% vs 36.1%; P = 0.07) or pregnancy loss (11.8% vs 16.1%; P = 0.16). A multivariable analysis found no impact of timing of elective FET on LBR (odds ratio, OR 0.73; 95% CI 0.49-1.08). The impact remained not significant after adjusting for number of retrieved oocytes, drug used for ovulation trigger (hCG vs GnRH agonist) and reason for cryopreservation. The factors that significantly affected LBR were: maternal age in both age categories (women between 35 and 40 years vs women below 35 years, OR 0.63, 95% CI 0.4-0.95; and women over 40 years vs women below 35 years, OR 0.34, 95% CI 0.2-0.7), day of embryonic development at transfer (day +4 vs +3; OR 1.7, 95% CI 1.1-2.8) and number of transferred embryos (OR 2.2, 95% CI 1.4-3.3) and oestrogen used for endometrial preparation (transdermal vs oral; OR 0.62, 95% CI 0.4-0.9). LIMITATIONS REASONS FOR CAUTION: The main limitation of our study is its retrospective nature. Although we adjusted our statistical analysis for a number of known and suspected confounders, we cannot exclude the possibility of residual confounding factors. WIDER IMPLICATIONS OF THE FINDINGS: According to our results, clinicians might not need to wait more than one menstrual cycle before performing FET. This allows us to reduce unnecessary delays in FET, without compromising reproductive outcomes. STUDY FUNDING/COMPETING INTERESTS: No funding was sought for this study. Authors declare no competing interests. TRIAL REGISTRATION NUMBER: NA.

PLK1 expression and BI 2536 effects in childhood acute lymphoblastic leukemia.

BACKGROUND: Polo-like kinase 1 (PLK1) is a conserved kinase that mediates various mitotic events. Compelling data have repeatedly demonstrated its upregulation in different neoplasia, being frequently associated with poor prognosis. However, in childhood acute lymphoblastic leukemia (ALL), no studies have yet been conducted. PROCEDURE: PLK1 expression and association with biological features were evaluated in 65 consecutively diagnosed childhood ALL samples by quantitative real-time PCR. Moreover, the effects of a specific PLK1 inhibitor, BI 2536, was tested against a panel of nine ALL cell lines at nanomolar concentrations (10, 50, 100 nM). RESULTS: The mRNA expression of PLK1 showed great variability in pediatric ALL, but no difference was evidenced compared to normal bone marrow. Additionally, no association was found between PLK1 mRNA expression with any clinical or biological features. Alternatively, high mRNA expression of PLK1 was present in ALL cell lines. In vitro treatment with BI 2536 strongly diminished growth, while presenting significant reduction in colony formation capacity and increased apoptosis rates. Moreover, strong G2/M arrest was detected suggesting important impaired proliferation after treatment. CONCLUSIONS: PLK1 mRNA expression level is not associated with prognosis in childhood ALL; however, considering the great variability observed in the sample and the in vitro experiments presented herein, BI 2536 treatment might serve as a promising therapeutic to enhance the efficacy of conventional treatment modalities in some childhood ALL cases.

Case report. Familial balanced translocation leading to an offspring with phenotypic manifestations of 9p syndrome.

We report two similarly affected cousins (children of monozygotic twin sisters) with phenotypic features consistent with 9p deletion syndrome, including dysmorphic craniofacial features (trigonocephaly, midface hypoplasia, upward-slanting palpebral fissures and long philtrum), intellectual disability and disorders of sex development. Initial cytogenetic examination showed normal karyotypes in the probands and their respective parents, though multiplex ligation probe amplification revealed a 1q terminal duplication and a 9p terminal deletion in both affected children. Further analysis by fluorescence in situ hybridization, identified a familial balanced cryptic translocation t(1;9)(q44;p23) in the mothers, showing the importance of the association of molecular cytogenetic techniques in clinical genetics, given the implications for the care of patients and for genetic counseling.

Osteosarcoma arising from osteochondroma of the tibia: case report and cytogenetic findings.

Osteochondroma is a cartilage capped benign tumor developing mainly at the juxta-epiphyseal region of long bones. The rate of malignant transformation, mainly into chondrosarcoma, is estimated to be less than 1-3%. Transformation into osteosarcoma is very rare and has been reported only thirteen times. There is little information on treatment and outcome. We report the case of a secondary osteosarcoma arising in the left tibia of a 23-year-old male, 10 years after the initial diagnosis of osteochondroma and after two partial resections. Malignant transformation occurred at the stalk and not at the cartilage cap, as would normally be expected. Chromosome banding analysis revealed the karyotype: 46,XY, t(3;13)(q21;q34) [2]/46,XY [18]. Records from additional cases will help determine the parameters that define these rare secondary bone lesions.

Tetraploidization in Wilms tumor in an infant.

Genetic instability is frequent in human cancer. Unscheduled tetraploidization can trigger cell transformation and tumorigenesis. We made a cytogenetic analysis by Giemsa-trypsin banding of a stage I, biphasic Wilms tumor diagnosed in a 10-month-old male. An evident karyotypic heterogeneity was found. Four different subclones of tumor cells were observed, with DNA content varying from diploid to near-tetraploid complements. The genetic events involved in the acquisition of aneuploidy in Wilms tumor remain unclear. We hypothesize that initial tetraploidization caused aberrant cell division, leading to abnormal chromosomal segregation, cell transformation and tumorigenesis.

Cytogenetic findings in an epithelioid sarcoma with angiomatoid features. A case report.

Epithelioid sarcoma is a rare, aggressive soft tissue tumor of unknown histogenesis showing predominantly epithelioid cytomorphology. We conducted a conventional and molecular cytogenetic study of a 27-year-old male with epithelioid sarcoma with angiomatoid features. Cytogenetic analysis of epithelioid sarcoma metaphase spreads by GTG-banding revealed a diploid chromosome complement with structural and numerical aberrations. Comparative genomic hybridization analysis demonstrated the amplification of 3p24-pter, 4p15.2-p16 and 18q23, while chromosome losses involved 3p13-p14, 3q24-q26.1, 9q21, and 11q21. Fluorescence in situ hybridization assessment showed normal hybridization patterns for the C-MYC and CCND1 loci; CCND1 RNA overexpression was detected by real-time polymerase chain reaction analysis. Genetic evaluation of this rare condition may be useful in determining if epithelioid sarcoma is associated with a distinct genetic background.

Downregulated Adhesion-Associated microRNAs as Prognostic Predictors in Childhood Osteosarcoma.

miRNAs have been identified as key regulators of almost all cellular processes, therefore, their dysregulation is involved with several diseases, including cancer. miRNAs specifically related to the metastastic cascade are called metastamiRs and can be involved with different steps of this process, including loss of adhesion. Osteosarcoma (OS) is the most common primary malignant pediatric bone tumor that often presents metastatic disease at diagnosis; therefore, a deeper study of adhesion-associated miRNAs could shed light on its pathophysiology. Online databases were used to select four miRNAs (miR-139; miR-181b; miR-584; miR-708) predicted or validated to target proteins related to adherent junctions and focal adhesion pathways, and their expression levels and possible associations with clinical features evaluated in primary OS samples. Our results showed downregulation of miR-139-5p and miR-708-5p in OS samples compared to non-neoplastic controls. Moreover, lower expression of miR-708-5p was associated with poor overall survival and higher expression of miR-181b-5p related to worst chemotherapy response (low HUVOS level). Based on these results, we selected miR-139-5p and miR-708-5p for further functional testing. Inducing the expression of miR-139-5p diminished the clonogenic capacity of the HOS cell line, while upregulation of miR-708-5p was related to a lower cellular adhesion. In summary, this work identified new signatures of microRNA dysregulation that may serve as useful prognostic markers in this aggressive pediatric bone tumor.

miRNA signatures in childhood sarcomas and their clinical implications.

Progresses in multimodal treatments have significantly improved the outcomes for childhood cancer. Nonetheless, for about one-third of patients with Ewing sarcoma, rhabdomyosarcoma, or osteosarcoma steady remission has remained intangible. Thus, new biomarkers to improve early diagnosis and the development of precision-targeted medicine remain imperative. Over the last decade, remarkable progress has been made in the basic understanding of miRNAs function and in interpreting the contribution of their dysregulation to cancer development and progression. On this basis, this review focuses on what has been learned about the pivotal roles of miRNAs in the regulation of key genes implicated in childhood sarcomas.

Leukemia/lymphoma-associated gene fusions in normal individuals.

Hematopoietic neoplasias are characterized by recurrent chromosomal aberrations that result in the formation of gene fusions and the subsequent expression of chimeric proteins with unique properties. However, in recent years, different lymphoma/leukemia-associated rearrangements, such as BCR/ABL, IGH/BCL2, ETV6/RUNX1 and MLL duplications, have been detected in healthy individuals. The presence of these rearrangements indicates that such translocations can be generated in normal hematopoietic cells without apparent oncogenic consequences. This article reviews and discusses the data available in the literature.

Control of the adsorption properties of alginate - guar gum matrix functionalized with epichlorohydrin through the addition of different flexible chain polymers as toll for the chymotrypsinogen isolation.

This work addresses the obtaining and characterization of alginate-guar gum matrix, cross-linked with epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinyl pyrrolidine and Pluronic® F68. These matrixes were used for the adsorption of chymotrypsinogen and showed an increasing uptake in presence of the flexible chain polymer in the sense: none < Pluronic 68 < polyvinyl pyrrolidine < polyvinyl alcohol. The adsorption process was found to follow a first order kinetics model and was not influenced by the polymer type. It was found that Freundlich model was more suitable for our data. Polyvinyl alcohol and polyvinyl pyrrolidine addition increase the adsorption capacity of the original bed due to an increment in the rigidity of the gel caused by the formation of hydrogen bound between the polysaccharides and synthetics polymers.

Comparison of soybean hull pre-treatments to obtain cellulose and chemical derivatives: Physical chemistry characterization.

The cellulose from soybean hull, a waste without value from the argentine agriculture, was successfully obtained by using two different treatments: the traditional alkaline-bleaching pathway and from a simple pre-alkaline treatment at low temperatures. The comparison of both methods yielded similar results regarding its ability to open the lignin cellulosic structure of the hull and the total elimination of the lignin content. Fourier Transform Infrared spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), 13C nuclear magnetic resonance (13C-RMN) and Raman spectroscopy were used to characterize the structures and the properties of cellulose. The results showed that cellulose can be easily obtained with just an alkaline pre-treatment of 5% (w/v) NaOH during 40 h at 50 °C and free of any lignin content. The attachment of different functional groups, such as -COOH and (CH3)3N+, changed the physicochemical properties of the obtained cellulose, showing mayor crystalline structure, and consequently modifying the swelling capacity and its ability to adsorb model proteins.

Analysis of ETV6/RUNX1 fusions for evaluating the late effects of cancer therapy in ALL (acute lymphoblastic leukemia) cured patients.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood. The improvements of therapies have increased the number of long-term survivors. However, an increased incidence of secondary neoplasias has been observed in this cohort. Our purpose was to evaluate the late effects of cancer therapy in cured patients previously treated for ALL, considering previous reports on the occurrence of gene fusions as putative markers of chromosomal instability. Twelve ALL patients (aged 5 to 16 years) and twelve healthy subjects (aged 18 to 22 years) were studied for the presence of ETV6/RUNX1 (TEL/AML1) translocations, which were detected by FISH (fluorescence in situ hybridization). The blood samples were collected months or years after completion of the therapy, and the frequencies of gene fusions in lymphocytes were compared with those obtained retrospectively for bone marrow samples at the time of diagnosis, and also for the control group. It was demonstrated that ETV6/RUNX1 gene fusion was a frequent event (0.59-1.84/100 cells) in peripheral blood lymphocytes from normal individuals and the ALL patients who underwent chemotherapy showed significantly (P = 0.0043) increased frequencies (0.62-3.96/100 cells) of the rearrangement when compared with the control groups (patients at diagnosis and healthy subjects). However, a significant difference was not found between the groups of patients at diagnosis and healthy subjects, when the two patients who were positive for the rearrangement were excluded. Therefore, increased frequencies of ETV6/RUNX1 fusions in ALL cured patients indicate the influence of previous exposure to anti-cancer drugs, and they may represent an important genetic marker for estimating the risk of relapse, or development of secondary neoplasias.

Prostaglandin levels in infertile patients affected by asthenozoospermia and prostatitis.

19-hydroxy-prostaglandins and prostaglandins of the E series (19-OH PGEs) were estimated in the seminal plasma of asthenozoospermic patients (n = 15) and individuals affected by prostatitis (n = 10) and compared to controls (n = 13) and secretory azoospermic patients (n = 8). All of them were free from infections (except individuals affected by prostatitis), biochemical and ultrastructural problems. The results indicate that endogenous prostaglandin levels (19-OH PGEs and PGEs) bear no correlation either to motility or absence of spermatozoa. Significant increases of PGEs were observed in patients affected with prostatitis. Surprisingly PGE levels showed no correlation with the levels of 19-OH PGEs.

Sperm recuperation and cervical insemination in retrograde ejaculation.

Seven infertile patients with retrograde ejaculation, in which spermatozoa could be recuperated from the postejaculation urine, were admitted to a sperm recuperation and cervical insemination program. A noninvasive method for sperm recuperation based on urine alcalinization and serial controls to time masturbation has been used. Insemination has been timed according to BBT charts and cervical mucus characteristics. Pregnancy has been obtained in the seven couples after one to eight treatment cycles.

Comparative cytogenetic studies of Curimatidae (Pisces, Characiformes) from the middle Paraná River (Argentina).

Almost all species of the Curimatidae family have a stable karyotype, with a diploid number of 54 metacentric (M) and submetacentric (SM) chromosomes, and one sole nucleolus organizer pair. This family has considerable specific diversity in Argentinean fluvial basins; however, no cytogenetic data are available. Eight species from the Paraná River (Argentina): Cyphocharax voga, C. spilotus, C. platanus, Steindachnerina brevipinna, S. conspersa, Curimatella dorsalis, Psectrogaster curviventris, and Potamorhina squamoralevis were analyzed cytogenetically. Chromosome preparations were obtained from direct samples and through cell culture, and they were processed for conventional, C- and nucleolar organizer region-banding. Six of the species exhibited the standard family karyotype, with 2n = 54 M-SM and fundamental number of chromosomes (FN) = 108, as well as variations in the chromosome formula, and in heterochromatic and nucleolar organizer regions. Though nucleolar organizer regions were located on only one chromosome pair, they varied in both carrier chromosomes and pairs involved. On the other hand, C. platanus showed a complement of 2n = 58 M-SM and subtelocentric with FN = 116, and P. squamoralevis presented 2n = 102, with some M-SM and a large number of acrocentric chromosomes. Even though the karyotype macrostructure appears to be conserved, the speciation process within the family has been accompanied by micro-structural rearrangements, as evidenced by pattern diversity in the heterochromatin and nucleolar organizer regions. Some changes in chromosome macrostructure have also occurred in this group, primarily in C. platanus and P. squamoralevis, in which there have been centric dissociations and inversions.

Inhibition of NF- κ B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and γ -Radiation Cytotoxicity in Glioblastoma Cells.

Despite advances in neurosurgery and aggressive treatment with temozolomide (TMZ) and radiation, the overall survival of patients with glioblastoma (GBM) remains poor. Vast evidence has indicated that the nuclear factor NF- κ B is constitutively activated in cancer cells, playing key roles in growth and survival. Recently, Dehydroxymethylepoxyquinomicin (DHMEQ) has shown to be a selective NF- κ B inhibitor with antiproliferative properties in GBM. In the present study, the ability of DHMEQ to surmount tumor's invasive nature and therapy resistance were further explored. Corroborating results showed that DHMEQ impaired cell growth in dose- and time-dependent manners with G2/M arrest when compared with control. Clonogenicity was also significantly diminished with increased apoptosis, though necrotic cell death was also observed at comparable levels. Notably, migration and invasion were inhibited accordingly with lowered expression of invasion-related genes. Moreover, concurrent combination with TMZ synergistically inhibited cell growth in all cell lines, as determined by proliferation and caspase-3 activation assays, though in those that express O(6)-methylguanine-DNA methyltransferase, the synergistic effects were schedule dependent. Pretreatment with DHMEQ equally sensitized cells to ionizing radiation. Taken together, our results strengthen the potential usefulness of DHMEQ in future therapeutic strategies for tumors that do not respond to conventional approaches.

Polo-like kinase 1 inhibition causes decreased proliferation by cell cycle arrest, leading to cell death in glioblastoma.

Glioblastoma (GBM) is one of the most aggressive central nervous system tumors with a patient's median survival of <1 year. Polo-like kinases (PLKs) are a family of serine/threonine kinases that have key roles in cell cycle control and DNA-damage response. We evaluated PLK1, 2, 3 and 4 gene expression in 8 GBM cell lines and 17 tumor samples, and analyzed the effect of the PLK1 inhibition on SF188 and T98G GBM cell lines and 13 primary cultures. Our data showed PLK1 overexpression and a variable altered expression of PLK2, 3 and 4 genes in GBM tumor samples and cell lines. Treatments with nanomolar concentrations of BI 2536, BI 6727, GW843682X or GSK461364 caused a significant decrease in GBM cells proliferation. Colony formation was also found to be inhibited (P<0.05), whereas apoptosis rate and mitotic index were significantly increased (P<0.05) after PLK1 inhibition in both GBM cell lines. Cell cycle analysis showed an arrest at G2 (P<0.05) and cell invasion was also decreased after PLK1 inhibition. Furthermore, simultaneous combinations of BI 2536 and temozolomide produced synergistic effects for both the cell lines after 48 h of treatment. Our findings suggest that PLK1 might be a promising target for the treatment of GBMs.