Oocytes evaluation and in-vitro fertilization/intra cytoplasmic sperm injection outcomes.

The identification of markers capable of evaluating oocyte quality, its maturation, function, and embryo progression and implantation potential has frequently initiated research interest. However, to date, univocal criteria of oocyte competence do not exist. A major cause of low oocyte quality is evidently advanced maternal age. However, other factors may influence oocyte competence. Among these are obesity, lifestyle factors, genetic and systematic pathologies, ovarian stimulation protocols, laboratory procedures, culture, and environmental conditions. The morphological and maturational evaluation of oocytes is probably the most widely used. Several morphological features, both cytoplasmic (cytoplasmic pattern and hue, presence of vacuoles, refractile bodies, granulation, and smooth endoplasmic reticulum clusters) and extra-cytoplasmic (perivitelline space, zona pellucida thickness, oocyte shape, and polar bodies), have been proposed to distinguish oocytes with the best reproductive potential among a cohort. No single abnormality seems to be sufficiently predictive of the developmental capacity of the oocyte. Some abnormalities such as cumulus cells dysmorphisms, central granulation, vacuoles, and smooth endoplasmic reticulum clusters, however, seem to be associated with poor developmental potential of the embryo, although oocyte dysmorphisms are very common and the data in the literature is limited and provide conflicting views. Other criteria involving gene expression of cumulus cells as well as the metabolomic analysis of spent culture media have been explored. Also, sophisticated technologies such as polar bodies biopsy, meiotic spindle visualization, mitochondrial activity, oxygen consumption, and measurement of glucose-6-phosphate dehydrogenase activity have been proposed. Many of these approaches, however, remain largely research-based and have not found widespread application in clinical service. Due to the lack of consistent data for the assessment of oocyte quality and competence, probably oocyte morphology and oocyte maturity remain important indicators to determine oocyte quality. The aim of this review was to provide spherical attributes and evidence on recent and present research on the topic by analyzing the current methods for evaluation of the oocyte quality, and the impact of oocyte quality on reproductive outcomes. Additionally, current limitations of oocyte quality evaluation are highlighted and insights on future research are provided to optimize the selection techniques of oocytes to improve ART outcomes.

Oocyte-triggering day progesterone levels and endometrial appearance in normoresponders undergoing IVF/ICSI cycles: a hypothesis and a study protocol.

In this report, we propose a study protocol capable of improving IVF outcomes in subfertile women with expected normal ovarian response. This proposal derives from conflicting published data and observations in our daily practice, concerning the negative impact of progesterone (P4) elevation at the day of oocyte triggering on pregnancy outcomes. Our hypothesis points to the combination of two previous "suspects" of reduced success after assisted reproduction techniques (ART) - the endometrium ultrasonographic parameters and P4 elevation at the day of oocyte triggering on their impact on pregnancy outcomes. Up-to-the minute data show that, there is a different impact of elevated P4 in fresh, frozen and donor cycles, whereas there are plenty of reports pointing to a different endometrial gene expression on different P4 measurements. Gaps in the literature are linked with a variation of the measurements of P4, its cycle-to-cycle reproducibility, the different cut-off levels used, the impact of various protocols of ovarian stimulation and the limitations of systematic reviews originating from the initial studies. Our hypothesis states that the combination of P4 values and endometrial ultrasound parameters at the day of oocyte triggering can affect clinical pregnancy rates in normal responders undergoing ART.

Estradiol Pretreatment in an Ultrashort GnRH Combined with a GnRH Antagonist Protocol in A Cohort of Poor Responders Undergoing IVF/ICSI: A Case-control Study.

AIM: To provide results of the use of estradiol pretreatment in a combination of an ultrashort gonadotropin-releasing hormone (GnRH) agonist and antagonist protocol, in an attempt to improve the clinical outcomes in "poor responders", according to the Bologna criteria, undergoing in vitro fertilization (IVF). PATIENTS AND METHODS: We applied estradiol pretreatment to 20 participants before the initiation of a combination of an ultrashort GnRH agonist plus an GnRH antagonist protocol followed by high doses of gonadotropins; the control group consisted of 20 subfertile participants with matching age, body mass index (BMI), basal follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH), antral follicle count (AFC) and cause of subfertility, conforming with the same inclusion criteria and treated with a fixed GnRH antagonist protocol. The primary outcome measure was live birth, while the secondary outcomes included embryological and cycle parameters. RESULTS: Live birth was determined in reduced rates in the study compared to the control group (0/20 vs. 3/20, p=0.231) as also in the respective number of clinical pregnancies (2/20 vs. 5/20, p=0.407) and cancellations (10/20 vs. 6/20, p=0.197), but none of these differences reached statistical significance. Also, most of the secondary parameters studied were similar for both groups. CONCLUSION: The study protocol does not seem to constitute an equally effective method compared to the GnRH antagonist protocol in the selected study population. The presented dissimilar results with those reported so far in the literature are possibly attributed to the small sample size and the strict criteria applied when labeling participants as "poor responders".

The Effect of Reactive Oxygen Species on Embryo Quality in IVF.

UNLABELLED: BACKROUND/AIM: Reactive oxygen species (ROS) are involved in critical biological processes in human reproduction. The aim of this study was to evaluate the association of embryo quality following in vitro fertilization (IVF), with ROS levels in the serum and follicular fluid (FF). MATERIALS AND METHODS: Eighty-five participants underwent ovarian stimulation and IVF; ROS levels were measured in blood samples on the day of oocyte retrieval and in the FF from follicular aspirates using enzyme-linked immunosorbent assay. These values were associated with the quality of embryos generated. RESULTS: Univariable zero-inflated Poisson model revealed that ROS levels at both oocyte retrieval and in FF were not associated with the number of grade I, II, III and IV embryos (p>0.05). Age, body mass index, stimulation protocol and smoking status were not associated with the number of embryos of any grade (p>0.05). CONCLUSION: Neither ROS levels in serum nor in FF are associated with the quality of embryos produced following IVF.

Leiomyomas and genomics.

Uterine leiomyomas represent a major health problem for women in reproductive age, as these benign monoclonal tumors introduce a pathological state in the female reproductive system structure and function. Despite their common clinical occurrence, the etiology of their incidence remains unclear. Several theories have been proposed in an attempt to clarify the etiology and route of tumor formation in leiomyomas, with estrogen/progesterone and several growth factors, cytokines, chemokines, genes and microRNAs to have been implied as key regulators in their growth. The structured theories presented so far indicate multiple candidates or an occurring interplay between these factors, with the reported findings to denote a composite molecular and biological involvement to attribute to the nature of their pathogenesis. Undoubtedly the evolving molecular technologies and recent developments allow a faster assembly of a vast array of data that should ultimately contribute in the establishment of the etiology and the related events leading to leiomyoma formation to ensure a more targeted management and treatment of the affected individuals.

In Vitro Maturation in Women with vs. without Polycystic Ovarian Syndrome: A Systematic Review and Meta-Analysis.

OBJECTIVE: To evaluate in vitro maturation (IVM) in sub-fertile women with polycystic ovarian syndrome (PCOS) undergoing in vitro fertilisation (IVF), by comparing outcomes with a control group of non-PCOS. STUDY DESIGN: A search strategy was developed for PubMed and studies reporting rates of the following outcomes (live birth; clinical pregnancy; implantation; cycle cancellation; oocyte maturation; oocyte fertilization; miscarriage) between patients with PCOS, PCO and controls undergoing IVM were deemed eligible. The review was conducted in accordance to the PRISMA guidelines and included studies quality was assessed through the Newcastle-Ottawa Quality scale. ORs with their corresponding 95% CIs were calculated for the main analysis and subgroup analyses were performed for PCOS cases vs. controls and PCOS vs. PCO cases. Alternative analyses were performed for live birth and clinical pregnancy, based on cycles and on women. Subgroup analyses for FSH stimulation, hCG priming and type of procedure (IVF/ICSI) were undertaken for all meta-analyses encompassing at least four study arms. Random effects models were used to calculate pooled effect estimates. RESULTS: Eleven studies were identified. A total of 268 PCOS patients (328 cycles), 100 PCO patients (110 cycles) and 440 controls (480 cycles) were included in the meta-analysis. A borderline trend towards higher birth rates among PCOS patients emerged (pooled OR = 1.74, 95%CI: 0.99-3.04) mainly reflected at the subgroup analysis vs. controls. Clinical pregnancy (pooled OR = 2.37, 95%CI: 1.53-3.68) and implantation rates (pooled OR = 1.73, 95%CI: 1.06-2.81) were higher, while cancellation rates lower (pooled OR = 0.18, 95%CI: 0.06-0.47) among PCOS vs. non-PCOS subjects; maturation and miscarriage rates did not differ between groups, while a borderline trend towards lower fertilization rates among PCOS patients was observed. CONCLUSION: The present meta-analysis provides preliminary evidence on the effectiveness of IVM as a treatment option when offered in sub-fertile PCOS women, as the latter present at least as high outcome rates as those in non-PCOS.

Review: MicroRNAs in assisted reproduction and their potential role in IVF failure.

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression stability. In the endometrium, miRNAs are involved in the dynamic changes associated with the menstrual cycle, implicated in implantation and in reproductive disorders. We performed a review in an attempt to assess the potential biological pathways linking altered miRNAs profiles with in vitro fertilisation (IVF) failure. Crucially, as miRNAs appear to have a significant role in the course of reproduction, they are excellent research candidates with the potential to enable a better understanding over the underlying molecular activities that prevent implantation and further progression of the embryo. Further steps include in-depth pathway mapping of the implantation process and the characterization of the respective miRNAs and associated links. The efficiency of any intervention should determine whether miRNA profiling could possibly be adopted in routine practice to substantially improve the diagnostic accuracy and, in parallel, the directed treatment of the next-generation IVF.

Transvaginal ovarian trauma, poor responders and improvement of success rates in IVF: anecdotal data and a hypothesis.

In this report, we propose an intervention capable of improving IVF outcomes in subfertile women with poor ovarian response. This intervention derives from anecdotal data and observations in our daily practice, but most importantly from trials on experimental models and subfertile women with Polycystic Ovarian Syndrome (PCOS). Our hypothesis suggests that transvaginal induction of trauma to the ovary in the cycle preceding IVF should benefit poor ovarian responders and their lowered pregnancy rates by increasing - at least - the number of retrieved oocytes during oocyte retrieval. Up-to-the minute data show that, via this means, there is a unique response of the ovarian surface epithelium and stroma to the induced trauma. The potential pathways of this beneficial response involve an improvement of the raised gonadotrophins to act either through the mechanical reduction of the size of the ovary or through alterations of the hormonal profile by lowering LH, inhibin and local androgen concentrations through hypothalamic-pituitary axis feedbacks, the induction of increased blood flow to the ovaries, a differentiated local immune reaction and a non-elucidated as yet role of reactive oxygen species. In this report, we also describe the technique and the associated possible negative points while we try to point out the needed research steps to ensure its efficiency before it enters daily clinical practice.

Pharmacologic suppression of JAK1/2 by JAK1/2 inhibitor AZD1480 potently inhibits IL-6-induced experimental prostate cancer metastases formation.

Metastatic prostate cancer is lethal and lacks effective strategies for prevention or treatment, requiring novel therapeutic approaches. Interleukin-6 (IL-6) is a cytokine that has been linked with prostate cancer pathogenesis by multiple studies. However, the direct functional roles of IL-6 in prostate cancer growth and progression have been unclear. In the present study, we show that IL-6 is produced in distant metastases of clinical prostate cancers. IL-6-activated signaling pathways in prostate cancer cells induced a robust 7-fold increase in metastases formation in nude mice. We further show that IL-6 promoted migratory prostate cancer cell phenotype, including increased prostate cancer cell migration, microtubule reorganization, and heterotypic adhesion of prostate cancer cells to endothelial cells. IL-6-driven metastasis was predominantly mediated by Stat3 and to lesser extent by ERK1/2. Most importantly, pharmacologic inhibition of Jak1/2 by AZD1480 suppressed IL-6-induced signaling, migratory prostate cancer cell phenotypes, and metastatic dissemination of prostate cancer in vivo in nude mice. In conclusion, we demonstrate that the cytokine IL-6 directly promotes prostate cancer metastasis in vitro and in vivo via Jak-Stat3 signaling pathway, and that IL-6-driven metastasis can be effectively suppressed by pharmacologic targeting of Jak1/2 using Jak1/2 inhibitor AZD1480. Our results therefore provide a strong rationale for further development of Jak1/2 inhibitors as therapy for metastatic prostate cancer.

Potential Pathophysiological Mechanisms of the Beneficial Role of Endometrial Injury in In Vitro Fertilization Outcome.

Successful embryo implantation is a complex process that involves multiple biological mechanisms and reciprocal interactions between the embryo and the proliferated endometrium. In this review, we provide an informative contribution on the pathways underlying the beneficial nature of endometrial injury toward improving implantation rates of embryos conceived and through in vitro fertilization. The evidence published to date are in favor of inducing local endometrial injury in the preceding cycle of ovarian stimulation to improve pregnancy outcomes in women with unexplained and recurrent implantation failure. Endometrial injury triggers a series of biological responses but the findings suggest that no particular pathway is solely adequate to explain the association between trauma and improved pregnancy rates rather than a cluster of events in response to trauma which benefits embryo implantation in ways both known and unknown to the scientific community.

STAT5A/B gene locus undergoes amplification during human prostate cancer progression.

The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.

A family with Hoyeraal-Hreidarsson syndrome and four variants in two genes of the telomerase core complex.

We describe an African American family with Hoyeraal-Hreidarrson syndrome (HHS) in which 2 TERT mutations (causing P530L and A880T amino acid changes) and two in the DKC1 variants (G486R and A487A) were segregating. Both genes are associated with dyskeratosis congenita and HHS. It was important to determine the importance of these mutations in disease pathogenesis to counsel family members. From genetic analysis of family members, telomere length and X-inactivation studies we concluded that compound heterozygosity for the TERT mutations was the major cause of HHS and the DKC1 G486R variant is a rare African variant unlikely to cause disease.

Efficacy of abiraterone acetate in post-docetaxel castration-resistant prostate cancer.

Evaluation of: Reid AH, Attard G, Danila DC et al. Significant and sustained antitumor activity in post-docetaxel, castration-resistant prostate cancer with the CYP17 inhibitor abiraterone acetate. J. Clin. Oncol. 28(9), 1489-1495 (2010). Inhibition of cytochrome P17 (CYP17), which is involved in androgen synthesis, is a promising therapeutic strategy for castration-resistant prostate cancer (CRPC). The first multicenter, Phase II study of a CYP17 inhibitor, the small molecule abiraterone acetate, has been conducted on 47 patients that received prior docetaxel chemotherapy for prostate cancer. With 1000 mg once daily, declines of more than 50% in prostate-specific antigen and circulating tumor cell counts were seen in 51 and 63% of patients, respectively. Overall, the drug was well tolerated and had a significant antitumor activity with symptomatic improvements. Reid and colleagues' study highlights abiraterone as a key molecule in CRPC treatment and gives further evidence of the involvement of the androgen receptor signaling axis in the disease. A randomized Phase III trial is ongoing to define the prognostic impact of this drug among the very limited arsenal of drugs currently available for CRPC. In the meantime, other CYP17 inhibitors are expected to show a favorable safety and efficacy profile, as are other novel powerful agents and combinatorial therapeutic strategies.

Stat5 promotes metastatic behavior of human prostate cancer cells in vitro and in vivo.

There are no effective therapies for disseminated prostate cancer. Constitutive activation of Stat5 in prostate cancer is associated with cancer lesions of high histological grade. We have shown that Stat5 is activated in 61% of distant metastases of clinical prostate cancer. Active Stat5 increased metastases formation of prostate cancer cells in nude mice by 11-fold in an experimental metastases assay. Active Stat5 promoted migration and invasion of prostate cancer cells, and induced rearrangement of the microtubule network. Active Stat5 expression was associated with decreased cell surface E-cadherin levels, while heterotypic adhesion of prostate cancer cells to endothelial cells was stimulated by active Stat5. Activation of Stat5 and Stat5-induced binding of prostate cancer cells to endothelial cells were decreased by inhibition of Src but not of Jak2. Gene expression profiling indicated that 21% of Stat5-regulated genes in prostate cancer cells were related to metastases, while 7.9% were related to proliferation and 3.9% to apoptosis. The work presented here provides the first evidence of Stat5 involvement in the induction of metastatic behavior of human prostate cancer cells in vitro and in vivo. Stat5 may provide a therapeutic target protein for disseminated prostate cancer.

Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics.

The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3-APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of conventional antibiotics including chloramphenicol, beta-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent.

HIF-1alpha and STAT3 client proteins interacting with the cancer chaperone Hsp90: therapeutic considerations.

Heat shock protein 90 (Hsp90) is an ATP-dependent chaperone and a noteworthy component of cellular folding machinery in eukaryotes and bacteria. What makes Hsp90 specifically promising as a target for anti-cancer drugs is that many of its client proteins are involved in signaling and chromatin-remodeling pathways, and these pathways are often disrupted in many types of cancers. A very recent study by Lang et al. (Clin Cancer Res 2007; 13:6459-68) provide compelling evidence supporting a novel IL-6/STAT3/HIF-1alpha autocrine loop in pancreatic cancer cells, emphasizing the arresting role of Hsp90 inhibitors. Here, we discuss up-to-date advances towards the development of novel Hsp90 inhibitors designed to selectively block the growth and proliferation of tumor cells and proving to be intriguing targets for current clinical studies and cancer therapeutics.

Following the tracks of AKT1 gene.

The AKT or PKB family of protein kinases is one of the best characterized targets of phosphoinositide 3-kinases or PI3Ks. The AKT/PKB signal transduction pathway regulates many growth and survival mechanisms including transcription, cell cycle progression, metabolism, apoptosis, invasion and metastasis. Recently, Carpten et al. (Nature 2007;448:439-44) reported a very interesting study on the isoform AKT1 (PKBalpha). They described a novel point mutation (E17K) in the pleckstrin homology domain (PHD) of the AKT1 gene in human breast, colorectal and ovarian cancers, and demonstrated that it induces leukemia in mice. Here we give a critical appraisal of this finding and underline its clinical impact. We also discuss possible interventions for therapeutic development of AKT inhibitors to treat human cancer.

Antiangiogenic drugs currently used for colorectal cancer: what other pathways can we target to prolong responses?

Angiogenesis plays a key role in facilitating tumor growth and metastasis of colorectal cancer. Considerable progress has been made in identifying molecular components to develop angiogenesis-based treatments. Vascular endothelial growth factor (VEGF) pathways are specific and critical regulators of angiogenesis. However, other pathways are indirectly involved in angiogenesis promotion and recent studies have confirmed it. This review discusses known mechanisms involved in the action of angiogenesis- related targeted drugs such as cetuximab (Erbitux) and bevacizumab (Avastin), in patients affected by colorectal cancer. It also elucidates the role of oxygen levels in cancer cell damage as well as known alternative pathways used by tumoral cells to escape hypoxic-related death. Hypoxia may in fact select resistant sub-clones and boost tumor progression and growth, while the main subsequent damages may be conferred by eventual re-oxygenation. Therefore, in the light of the consolidated data available from the use of antiangiogenic drugs, we discuss about a paradox that is forming: in the war against tumoral angiogenesis it will be necessary to fight also the hypoxic condition caused by the antiangiogenic drugs.

Helicobacter pylori as a class I carcinogen: physiopathology and management strategies.

The gram-negative bacterium Helicobacter pylori is known as a persistent colonizer of the human stomach, and probably less known is that it is also involved in extraintestinal diseases. Public awareness of its contribution in the development of gastric cancer is less than 15 years old. The efficacy of the current therapies based on antibiotics against H. pylori has been limited by difficulties such as antibiotic resistance and recurrence. As a consequence, the development of promising vaccines was prompted as the best preventive measure. Unfortunately, so far vaccines failed the transition from animal models to human trials. This keynote presentation is to provide a bird's eye view of H. pylori-related gastric diseases, including gastric cancer, with a synthesis of the molecular mechanisms involved, and an exhaustive presentation and discussion of the current therapeutic guidelines and future strategies for prevention or therapy.