Analyzing differential-gene-expression data in the context of protein-interaction networks (PINs) yields information on the functional cellular status. PINs can be formally represented as graphs, and approximating PINs as undirected graphs allows the network properties to be characterized using well-established graph measures.This paper outlines features of PINs derived from 29 studies on differential gene expression in cancer. For each study the number of differentially regulated genes was determined and used as a basis for PIN construction utilizing the Online Predicted Human Interaction Database.Closeness Centrality, Graph Diameter, Index of Aggregation, Assortative Mixing Coefficient, Connectivity, Sum of the Wiener Number, modified Vertex Distance Number, and Eigenvalues differed clearly between PINs derived on the basis of differential gene expression data sets characterizing malignant tissue and PINs derived on the basis of randomly selected protein lists.Graph measures calculated for the largest subgraph of a PIN for a given differential-gene-expression data set comprised properties reflecting the size, distribution, biological relevance, density, modularity, and cycles. The values of a distinct set of graph measures, namely Cancer PINs representing differentially regulated genes are larger than those of randomly selected protein lists, indicating functional dependencies among protein lists that can be identified on the basis of transcriptomics experiments. However, the prevalence of hub proteins was not increased in the presence of cancer. Interpretation of such graphs in the context of robustness may yield novel therapies based on synthetic lethality that are more effective than focusing on single-action drugs for cancer treatment. The "omics" revolution has dramatically increased the amount of data available for characterizing intracellular events at the cellular level. The main experimental methodologies responsible for this development have included differential gene expression analysis for recording mRNA concentration profiles, and proteomics for providing data on protein abundance ,2. Each We have recently outlined a computational analysis workflow aimed at characterizing cellular events at a functional level, which includes the use of differential gene expression and proteomics data, analysis of transcriptional control, and coregulation via joint transcription factor modules, further complemented by protein interaction and functional pathway data . A majorThere are several experimental techniques for directly addressing protein-protein interactions, with the yeast two-hybrid system being the most commonly used . The yeaPublic-domain databases have been established for making protein-protein-interaction data readily accessible. The Online Predicted Human Interaction Database (OPHID) is a collection of human protein-protein interactions assembled from other databases and complemented by homolog interactions identified in other organisms . The OPHInformation on pairwise protein interactions as provided by the OPHID can be used to delineate protein interaction networks (PINs), which are usually represented as undirected graphs. Routines have been published for automatically generating and visualizing such interaction graphs ,14, wherLike many real-world networks, biological networks are scale-free in nature, with the majority of nodes showing a low degree of connectivity, complemented by some highly connected nodes serving as hubs ,17. The Various groups have applied network analysis to gene data sets associated with cancer. Jonsson and Bates reported very recently that proteins associated with cancer show an increased number of interacting partners in the interactome, reflecting their increased centrality in the PIN . Wachi eThe present paper provides a systematic analysis of properties computed for PINs represented as graphs, as exemplified by an extensive set of differential gene expression profiles covering various tumors. The primary hypothesis was that differential gene expression analysis provides systematic data on concerted events in malignant tissue , and theThe formal representation of PINs as undirected graphs makes it possible to utilize a variety of well-established graph measures. Junker and colleagues recently presented a tool for exploring centralities in biological networks, named CentiBiN . CentiBiWe have computed 22 individual graph measures for 29 tumor-associated differential gene expression data sets that reflect the following graph properties: size, distribution, relevance, density, modularity, and cycles. These graph measures provide a detailed characterization of the differential gene-expression data represented at the level of protein interactions.N), the number of nodes in graph (G), as well as the number of nodes in the largest subgraph (G') for the 29 studies. Furthermore, the characteristics of the individual studies as included in the Oncomine database [A mean of 90 genes  were identified as significantly differentially regulated for each transcriptomics experiment, and these genes were selected for constructing the entire graph for each given data set. Table database  are listG (after performing the nearest-neighbor expansion) was 140  for the 29 studies, with a mean of 109 nodes for the largest subgraph G' . For seven of the studies there were less than 30 nodes in the largest subgraph. Measures related to size, distribution, biological relevance, density, modularity, and cycles were computed for each subgraph G'.The mean number of nodes in Closeness Centrality, Graph Diameter, and Index of Aggregation – were different for networks generated from gene lists derived from Oncomine than for randomly generated protein lists  differed between Oncomine and random data sets , the total number of nodes in graph G , and the total number of nodes in the largest subgraph G'  revealed the dependence on number of nodes selected and the degree of deviation from random selections. This correlation was significantly affected by the small graphs analyzed, since studies resulting in subgraph sizes of less than 10 do not provide conclusive graph measures.To study the data sets at the level of the graph-measure categories, the 22 graph properties of each data set were checked for measures that significantly deviated from those of random graphs. Results of this evaluation are listed in Table r2 = 0.09, p = 0.12), nor with the number of graph measures deviating from the randomly generated reference sets .Interestingly, the number of samples analyzed for differential gene expression was not significantly correlated with the number of statistically significant differentially regulated genes found  between cancer networks and networks based on randomly generated data sets were Index of Aggregation was on average higher in tumor networks, indicating dependencies between proteins involved in cancer, as also proposed by Chen et al. in the context of Alzheimer disease [Graph Diameter and Closeness Centrality were significantly lower in tumor networks. This finding was also reported by Yu and colleagues for networks solely including highly expressed genes in the yeast interactome [Closeness Centrality values for tumor networks may initially appear surprising, but relative large size of the largest subgraphs in tumor networks  makes higher Closeness Centrality values harder to obtain. The largest subgraph of tumor networks also more elongated shortest paths between nodes.All three measures associated with the size of the graph differed significantly between tumor networks and randomly generated networks. The  disease . This in disease . Howevereractome . Low CloAssortative Mixing Coefficient, differed significantly in tumor networks. This coefficient is influenced by both the number of hub proteins and the number of edges, and a large number of hub proteins is correlated with an unequal distribution in the number of edges. The Assortative Mixing Coefficient is directly proportional to the number of edges and inversly proportional to the number of hub proteins. According to Jonsson and colleagues, tumor networks contain numerous hub proteins [One measure of the distribution category, the proteins . HoweverSum of the Wiener Number characterizes the density of the graph. The significantly higher values of this measure in tumor networks indicate larger graphs, which is consistent with the observed Index of Aggregation. We found that the Connectivity was lower in the largest subgraphs of tumor networks. This may be also due to the largest subgraphs of tumor networks being on average larger than the subgraphs of randomly generated gene lists, corresponding to low values of Closeness Centrality.The modified Vertex Distance Number is also influenced by the sum of shortest paths between two vertices, but in contrast to Closeness Centrality, all vertices in the OPHID network are considered. A higher modified Vertex Distance Number in tumor networks indicates higher connectivity and modularity in Oncomine networks. Finally, higher Eigenvalues values indicate the presence of fewer cycles in tumor networks.The Our analysis of 29 studies on differential gene expression in cancer has revealed a general tendency toward large subgraphs without the presence of explicit hubs. Comparing the graph measures between the individual gene expression studies and randomly selected genes provided a heterogeneous picture. Gene-expression studies resulting in a low number of statistically significant differentially regulated sequences  do not support an interpretation at the level of PINs , as resulting from the analysis of differential-gene-expression data.We used Oncomine as a central repository for differential-gene-expression data . This daWe extracted each file and processed the raw data according to the following scheme: The two groups per study were analyzed at the level of individual genes by computing a probability value for the differential expression of a particular gene in that given experiment. Multiple testing was accounted for by using the Holm-Sidak step-down test and setting the significance level to 0.05 . This prEach of the 29 selected differential gene expression studies was represented by a list of genes exhibiting significant differential regulation when comparing expression values for the group of tumor samples and the group of reference samples. Each gene on these lists was represented by its Gene Symbol, allowing a direct match with the protein interaction data as derived from the OPHID.G) were constructed for each gene list of the 29 selected gene-expression studies based on OPHID interaction data utilizing the nearest-neighbor expansion. This procedure built edges between the nodes of entries A and B of a given gene list if the interaction between A and B was directly encoded in the OPHID, or if one element X was identified in the OPHID, allowing the construction of an interaction of the type A - X - B, where X was not listed in the gene expression data set [Protein interaction graphs (data set .G comprising n subgraphs G' was constructed on the basis of genes in the initial list and their nearest neighbors in the PIN. G' is defined as a graph whose vertices and edges form subsets of the vertices and edges of G.For each gene list, entire graph G' was generated again following the nearest-neighbor expansion as outlined above, and the graph measures were computed for each G'. This procedure yielded the mean value and 2.5% lower and upper confidence limits for each graph measure for each data set size represented by the 1000 individual data sets.Gene lists derived from analyzing differential gene expression might be linked on the level of coregulation and protein interactions. To quantitatively assess such dependencies, the graph properties of PINs derived on the basis of randomly selected gene lists were computed as follows: Proteins encoded by randomly selected gene lists exhibit a background level of protein interactions, and we analyzed graph measures characterizing gene expression data sets with respect to random data sets. One thousand random gene sets containing between 10 and 300 genes were picked in steps of 10. For each of these gene sets, the largest subgraph G' were then determined for each Oncomine data set as well as for random data sets. Table The graph measures for each largest subgraph BM and PP designed the study. AP extended the concept, developed the software, and performed all the calculations. AP, PP, AL, and BM contributed to data interpretation and writing the manuscript. All authors read and approved the final manuscript.


Journal clubs are an internationally recognized teaching tool in many postgraduate medical education fields. In developing countries lack of funds for current print materials may have limited journal club use. But with advancing information technology trainees in developing countries increasingly have more access to high quality journals online. However, we are aware of no studies describing journal club existence and effectiveness in postgraduate medical training in Pakistan. Also we have found no published effectiveness studies of this teaching modality in Community Medicine  in any country. This study evaluated the effectiveness of Community Medicine  Resident Journal Club (CMR-JC) in Aga Khan University, Pakistan using international criteria for successful journal clubs (2 years continuous existence and more than 50% attendance) and examining resident and alumni satisfaction.Journal club effectiveness criteria were searched using electronic search databases. Departmental records were reviewed from September1999–September 2005. Ninety percent of residents and alumni of Community Medicine Residency Programme participated voluntarily in a confidential survey.The CMR-JC was regularly conducted. More than 95% of residents attended. . Twenty-seven out of 29 current residents/alumni responded to the anonymous questionnaire. Acquisition of critical appraisal skills (23 respondents) and keeping up with current literature (18 respondents) were the two most important objectives achieved. Respondents recommended improved faculty participation and incorporating a structured checklist for article review.CMR-JC fulfils criteria for effective journal clubs. Residents and alumni agree CMR-JC meets its objectives. Incorporating suggested recommendations will further improve standards. The journal club learning modality should be included in residency training programs in developing countries. Effective use of online resources to support journal clubs is demonstrated as a successful alternative to excessive expenditure for obtaining print journals. Those trying to start or improve journal clubs can benefit from our experience. A journal club can be defined as a group of individuals who meet regularly to discuss articles in current medical literature . The earAt the start journal clubs helped students stay current with medical literature. Later, they improved acquisition of knowledge in clinical epidemiology, biostatistics, research design, and more recently in teaching critical appraisal skills -8. StudiMany clinical disciplines report using journal clubs to train postgraduate trainees in relevant specialties. At present journal clubs are found in Medicine , Surgery , Psychiatry, Geriatrics, Nursing, and Health Care Management ,20-29. BWhile a 1999 article from the Royal College of Physicians London illustrates the popularity of journal clubs in public health medicine related to clinical practice,  the exisIn order to find criteria of effective journal clubs, articles were identified through electronic searches using PubMed, and Cochrane retrieval systems. Search terms used were "journal club", "effectiveness", "public health" and "post graduate medical education". Reference lists of known systematic reviews were also searched in this connection besides hand search of library periodicals ,32-36.The following criteria and associated factors of effective journal clubs were found:- More than 2 years of existence without periodic abandonment- More than 50 percent attendance of the expected audience- Explicit written learning objectives- Having a designated club leader- Mandatory attendance- Formal teaching of critical appraisal skills- Journal club independent of faculty journal club- Regular attendance by faculty- High value given by program director- Smaller residency programme (12 or less residents)- Incorporation of adult learning principles- Provision of free food- Use of a structured checklist for article reviewThe purpose of this study was to determine the effectiveness of the CMR-JC currently conducted in a Pakistan medical university by:1) Comparing the criteria of effective journal clubs in the international literature with that of the CMR-JC2) Seeking evidence of resident and alumni satisfaction with CMR-JCCMR-JC is offered by the Community Medicine Residency Program  of a Pakistan medical university. The target audience is Community Medicine Residents . These trainees already have a MBBS degree.The goals of CMR-JC are to:• increase awareness of residents regarding important national and international public health issues• teach residents critical appraisal skills• meet core competencies required by the College of Physicians and Surgeons Pakistan, the program accreditation body• assess residents' knowledge and expertise over time in raising public health issues, utilizing public health tools and developing strategies to address issues.The CMR-JC began in September 1999. It is conducted once every week for one hour. Attendance is mandatory for all Community Medicine Residents but students and faculty from other departments may also attend. On average there are 12 residents in the program, thus each resident is required to present every twelfth week. A presentation is followed by an interactive discussion. After the session, the presenting resident hosts a breakfast for participants.Senior residents of the program act as journal club coordinator on a rotational basis. Their responsibilities include helping other residents in article selection, preparing presentations, organizing mock presentations before the actual presentation day, ensuring the selected article reaches every one in the department at least two days prior to the meeting, and maintaining a record of journal club presentations. The journal club coordinator (resident) is guided by the coordinator and director of residency program.The residents choose original articles of public health importance published within the past three years in indexed journals. Residents can also present their research work and experiences of placement done or training attended. Sometimes alumni of the program are invited to share important research they have conducted.The resident first presents the rationale for choosing a particular topic by summarizing a background search indicating the global and local burden of the problem. Then the article is discussed according to Introduction, Methodology, Results, Discussion and Conclusion. Finally, a critique of the article is presented. The journal club coordinator facilitates discussion of strengths and weakness of the study. The issue is then related to the local context and possible solutions discussed. Residency faculty supervises the session and provides feedback.The presenting resident is evaluated by the faculty and senior residents on a standard evaluation form containing ten areas including the degree to which problem background relevant to Pakistan was provided, whether the resident was able to apply the article's information to a relevant context, and whether responses to queries were answered satisfactorily. Evaluations are compiled quarterly and are included in the overall evaluation of the resident.The primary outcome measures for this study were:1. To check success of CMR-JC through 2 success criteria reported in the literature (i.e. continuous existence for 2 years or longer and an estimated attendance of 50% or higher)2. To check through a survey whether the alumni and residents are satisfied with CMR-JC activity.Hence the study included two stages; the first during which a review of the format and characteristic of CMR-JC encompassing the period from September15, 1999 to September 15, 2005 was carried out. This was done to match for the above mentioned criteria for effective journal clubs coming from the literature search. The Second stage comprised a cross-sectional survey for assessment of participant satisfaction with the CMR-JC  and placement presentations. The data collected were verified by the alumni, faculty and residents of the CMR Program for accuracy and completeness. We reviewed the characteristics of the CMR-JC with regard to presence or absence of the earlier mentioned criteria and associated factors of successful journal clubs as well as annual number , type , name of the journal in which the article was published and topic area of presentations . All of these presentations and articles were in English.An anonymous questionnaire was mailed to the alumni working outside the department and circulated directly to current residents. Inclusion criteria encompass all current residents; all alumni; all who did not complete residency but who have attended CMR-JC for at least one year and have presented at least thrice in the forum. Since the start of CMR-JC, a total of 32 residents have gone through the residency program and we were able to contact more than 90% of them. Of the 29 resident/alumni contacted, 27 responded to the structured questionnaire.th "others" option. These questions were pertaining for example to the preferred method of continuing education, important reasons for attending CMR-JC and opinion regarding mock (practice) presentations and journal club coordination. Data entry and analysis was carried out using Microsoft Excel (version 2003) using standard statistical analysis.The questionnaire contained 23 questions.  are mentioned in table Four residents each were from first and fourth years and 2 residents were in second year. At the time of survey there was no third year resident in the program. Fifteen of the respondents were alumni of the residency program. Interestingly, more than 10% of the respondents were those who have not completed their residency but have responded to the survey questionnaire. Twelve (42.3%) of the respondents were female.The participants rated journal club (33.3%) as the second most preferred method of continuing education besides courses (37.0%) offered by the department. Learning of critical appraisal skills (44.4%), keeping up with current literature (29.6%), and requirement of mandatory attendance (21.4%) were cited as the most important reasons for attending CMR-JC.The questionnaire asked the respondents to assess themselves in the areas of knowledge (keeping up with current literature), skills  and behaviour (enhancement in reading habits as a result of participating in CMR-JC) as described in Figure Table We examined effectiveness criteria of successful journal clubs from various residency programs across the world and compared these with CMR-JC. Our study identified that both of the effectiveness criteria were present in CMR-JC.We also matched CMR-JC with the factors associated with effectiveness. All but one associated factors of effective journal clubs were found in CMR-JC and this was the use of standard check list for article review. In addition, faculty participation was one attribute which was found partially in CMR-JC Table .Studies have found that journal clubs benefit from an explicit expectation that residents attend journal club and if journal clubs are highly valued by the program director ,37,38. RProvision of food is associated with long-term, continuous existence of journal clubs . Many clresidency program faculty participation is always there yet one of the associated attributes of successful journal clubs which is found partially in CMR-JC is faculty participation i.e. the faculty members of the department who are not part of Community Medicine Residency Program attend CMR-JC sporadically. This may account for why the study found participants dissatisfied that fewer departmental faculty outside the residency program attend the CMR-JC .Good public health decisions depend on combining good information (routinely collected statistics) with good research evidence . FurtherThis study shows that a journal club can help residents develop the habit of staying updated on the significant literature that is published every year in medical science. Against the backdrop of a general resource constraint in developing countries, CMR-JC has made effective use of information technology for selecting articles from reputed international journals online. Given the free access to 17,000 scientific journals online, by Higher Education Commission (HEC) of Government of Pakistan; public sector medical universities can utilize the approach of journal club to train postgraduate trainees to make their practice evidence based . This exHaving shared the experience of a journal club which has thrived for more than six years and is popular among its participants, we would like to recommend that CMR-JC has shown a format for organizing and successfully running journal clubs in a developing country's setting with resource constraints and limited access to print publications. This design can be used by any residency program in similar settings for teaching and assessing resident's ability to critically appraise research as a core competency. Moreover this format can also be used for assessing the overall efficacy of journal clubs in meeting their objectives.Currently in Pakistan the existence and effectiveness of journal club meetings in postgraduate medical education is unreported. We achieved greater than 90 percent response rate, yet the sample represents only one medical university from Pakistan. Therefore, generalization requires caution. We recommend the study be replicated in other public health residency programmes in Pakistan and internationally. The literature also suggests that regular assessment surveys of journal clubs are recommended and we support this recommendation.A majority of participants in this study have recommended improved faculty participation in the journal club. It would be interesting and helpful to further explore what residents would like to see regarding faculty participation. It would also be valuable to look at faculty perspective regarding participation in CMR-JC.While this study has particular relevance for Pakistan, continued assessment of journal clubs, enhanced by the learning from this study, can contribute to public health-related journal club establishment and assessment wherever public health practitioners are trained.The author(s) declare that they have no competing interests.SA participated in the design of the study, collected and analyzed data and drafted the manuscript. MMK conceived the idea, supervised the study and gave intellectual input in the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Survey Questionnaire. Survey questionnaire sent to the residents and alumni of Community Medicine Residency Program to meet the second objective of the study.Click here for file


Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development. Sperm morphology is regarded as a significant prognostic factor for fertilization and pregnancy . AbnormaAtomic force microscopy (AFM) has opened up new avenues of study in reproductive biology. AFM, invented by Binnig, Quate and Gerber in 1986, has evolved as a powerful imaging technique to obtain nanometer-resolved topographic data images. In brief, the sample surface is raster scanned by a flexible cantilever with a sharp tip at one end. A laser beam focused on the back of the cantilever is bounced off and is detected by a photodiode detector. The ability of this technique to image non-conductive living cells in physiological environment (aqueous solution) in 3D array without elaborate sample preparation or fixing of samples unlike conventional electron microscopy  has made AFM a valuable tool to study various biomolecules -4, incluAFM provides the advantage of directly observing spermatozoa in their native environment thereby opening the exciting possibility of analyzing their structural and functional aspects at the sub-molecular level. This article provides a review on morphological and topological images of sperm cells using AFM. Such high-resolution images are expected to provide a better understanding of male factor infertility, improve the success rate of ART procedures and also give a new direction towards contraceptive development.Figure AFM has been used for morphologic and morphometric analyses of acrosome intact and acrosome-reacted human sperm heads . StructuMorphological alterations in spermatozoa leading to oligoasthenoteratozoospermia (OAT) and asthenozoospermia have been analyzed using AFM . This stDetailed topology of bovine spermatozoa and force vs. distance curves has been obtained using contact mode AFM . The acrAxoneme, a specific "9 + 2" arrangement of the microtubules in which nine outer doublet microtubules surround a central pair of singlet microtubules, plays an important role in the movement of spermatozoa. The bending of cilia and flagella is attributed to dynein-induced sliding of microtubules, which is a key step in force generation. A transverse function of the microtubule is predicted on the basis of the 3D movement of dynein motors i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. This has been confirmed using optical trapping  and elecE. cirrhosa has been studied in great detail [Xenopus sperm chromosomes has been suggested [DNA, present in a highly condensed state in mammalian sperm cells, was imaged successfully for the first time in both, air and liquids by Allen et al. (1993) . The higt detail . A simpluggested . Artefacuggested . The potSmithopsis crassicaudata and Trichosurus vulpecula has been carried out using AFM [Protamines, small arginine-rich protein, are the major DNA-binding proteins in the nucleus of spermatozoa of most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. The binding of protamine to sperm chromatin generates a large dense hydrophobic complex making the sperm chromatin structure difficult for microscopic examination. AFM imaging of the well-spread isolated sperm nuclei subjected to prior hypotonic treatment showed large nodular structures and a smaller nucleosome like particle near the periphery of the nucleus present in the chromatin . Similarsing AFM . The ressing AFM . The comAFM has been used to perform volume measurements of the human sperm nuclei by Lee et al. (1997) . Their rConstant force applied on the soft biological samples may damage the cell and thus change its morphology. Considering this, various imaging modes have been developed such as resonance based tapping mode, lift mode, force modulation imaging, nanoindenting, scratching and lateral force microscopy. The development of small micro-mechanized cantilever and optical fiber tips reduce thermal noise by providing a better ratio of cantilever stiffness and resonance frequency and improved imaging bandwidth. An important development would be the construction of antibody modified tips which could be useful in localizing antigens  on the plasma membrane. This may be helpful in studying molecular interactions in greater detail.AFM, in itself, has proved to be a powerful instrument in nanoscopic analysis of biological samples. Nevertheless, more information with finer details may be achieved if combined with various other techniques such as optical microscope  and optiTime lapse AFM imaging has been used to observe the conformational changes in supercoiled DNA  and in cP. falciparum merozoites [Scanning Near-field Optical Microscope (SNOM) is an emerging technique and is still in infancy with respect to the imaging of biological cells. This technique utilizes the near field, non-propagating component of light to scan the samples with optical tips, which can be applied in the tapping or contact mode. A resolution of few tens of nanometers is achievable by SNOM. High resolution topographic and optical images of sea urchin sperm flagellum have been obtained using fluorescent probe as a light . Electrorozoites . This meRecent developments in AFM have made it a powerful tool for analyzing biomolecules. Over the last decade, AFM has emerged as a valuable technique with extensive applications in the field of sperm biology. AFM is expected to provide a better understanding of various biological pathways and intermolecular interactions which will open up new avenues in reproductive medicine.SK has contributed to the acquisition of a part of the data by carrying out AFM experimental studies on spermatozoa treated with the contraceptive, RISUG. PS has also assisted in acquiring data and analyzing it. In addition, he has done extensive literature survey and collected the data/research papers. KC has conceived the study and contributed to the design, analysis, co-ordination and interpretation of data. SKG, the inventor of RISUG, has also participated in the design, revised the manuscript critically for important intellectual content and has given final approval of the version to be published. All authors read and approved the final manuscript.


Heart failure is the most common cause of hospitalization among adults over 65. Over 60% of patients die within 10 years of first onset of symptoms. The objective of this study is to determine the effectiveness of self-management interventions on hospital readmission rates, mortality, and health-related quality of life in patients diagnosed with heart failure.The study is a systematic review of randomized controlled trials. The following data sources were used: MEDLINE (1966-11/2005), EMBASE (1980-11/2005), CINAHL (1982-11/2005), the ACP Journal Club database (to 11/2005), the Cochrane Central Trial Registry and the Cochrane Database of Systematic Reviews (to 11/2005); article reference lists; and experts in the field. We included randomized controlled trials of self-management interventions that enrolled patients 18 years of age or older who were diagnosed with heart failure. The primary outcomes of interest were all-cause hospital readmissions, hospital readmissions due to heart failure, and mortality. Secondary outcomes were compliance with treatment and quality of life scores. Three reviewers independently assessed the quality of each study and abstracted the results. For each included study, we computed the pooled odds ratios (OR) for all-cause hospital readmission, hospital readmission due to heart failure, and death. We used a fixed effects model to quantitatively synthesize results. We were not able to pool effects on health-related quality of life and measures of compliance with treatment, but we summarized the findings from the relevant studies. We also summarized the reported cost savings.From 671 citations that were identified, 6 randomized trials with 857 patients were included in the review. Self-management decreased all-cause hospital readmissions  0.44 to 0.80, P = 0.001) and heart failure readmissions . The effect on mortality was not significant . Adherence to prescribed medical advice improved, but there was no significant difference in functional capabilities, symptom status and quality of life. The reported savings ranged from $1300 to $7515 per patient per year.Self-management programs targeted for patients with heart failure decrease overall hospital readmissions and readmissions for heart failure. It is estimated that as many as 30 million of the one billion people living in the 47 nations represented by the European Society of Cardiology are living with heart failure (HF) . The estDisease management programs can reduce hospitalization and may improve the quality of life of patients diagnosed with heart failure -10. CompSelf-management strategies have proven beneficial in chronic diseases such as asthma and type 2 diabetes mellitus ,12, but We searched MEDLINE from 1966 to November 2005, EMBASE from 1980 to November 2005, CINAHL from 1982 to November 2005, the ACP Journal Club database, the Cochrane Central Trial Registry and the Cochrane Database of Systematic Reviews. The search strategy used the terms: "self management", "self administration", "self medication", "quality of care", "quality of healthcare", "disease management", "congestive heart failure" and "heart failure, congestive". We limited the studies to randomized controlled trials using the terms "randomized controlled trial", "controlled clinical trial", "random allocation" and "random*". We placed no restrictions on languages. We identified additional articles from reviewing the bibliographies of retrieved articles and from consulting experts in the field. Further details on the search strategies are available from the authors.We included randomized controlled trials of self-management interventions that enrolled patients 18 years of age or older who were diagnosed with heart failure. The target population consisted of patients hospitalized for heart failure who were enrolled in self-management programs during the hospitalization or at discharge.Health Canada defines self-management as "decisions and actions taken by someone who is facing a health problem or issue in order to cope with it and improve his or her health" . The opeThe primary outcomes of interest were all-cause hospital readmissions, hospital readmissions due to heart failure and mortality. Secondary outcomes were compliance with treatment, adherence to self-management strategies and quality of life scores. We included studies that collected data relevant to the primary and secondary outcomes, regardless of the measure of hospital readmission. Since we were primarily interested in determining the effects of the intervention on readmission to hospital and mortality, we excluded studies that enrolled the patients in the community setting.Two reviewers  independently reviewed the abstracts obtained in the search and retrieved the full text article of those that met the inclusion criteria. In cases of disagreement, we retrieved the full text article for review. Three reviewers  independently reviewed all retrieved articles to confirm that the inclusion criteria were met and to assess the quality of each study. The quality assessment criteria included the method of randomization, details of allocation concealment, blinding of health care provider, outcome assessors and data analysts, intention-to-treat analysis and method of outcome assessment. The reviewers resolved differences in assessment through discussion to achieve consensus.Three reviewers  independently extracted data on the details of the intervention and the outcomes of interest from all studies selected for inclusion in the systematic review. The reviewers resolved differences in assessment through discussion to achieve consensus.For each study, the authors computed the pooled odds ratios (OR) for dichotomous variables including all-cause hospital readmission, heart failure readmission and death of patients in the intervention and control groups. Statistical heterogeneity was assessed using the Q-test . A fixedThe search strategies yielded 642 articles. We identified an additional 29 articles from personal files, reference lists of retrieved articles and from communication with experts. Based on two independent reviews of the abstracts, we retrieved the full text of 65 articles. The agreement on the reviewers' initial assessment on whether to include or exclude the articles these articles was 89% (58 articles of the 65 articles retrieved). After excluding articles that did not meet the inclusion criteria and removing duplicates, we identified six studies of sufficient quality for inclusion in the systematic review. All excluded studies, except for one, were excluded because they either did not meet the definition of self-management or because their results were published in more than one study. One self-management study was excluded because it was conducted in a community setting.Table All studies but one  consisteLength of follow-up varied across the trials, ranging from three to twelve months. All studies reported rate of hospital readmission as a primary or secondary outcome. Four of six studies reported mortality rates at one year. Three of six studies reported scores for patients' quality of life. Three studies reported effects on patients' health behavior.P = 0.93). Pooling of these results identified a significant decrease in hospitalizations with self-management . The effect remained unchanged after the study that did not clearly specify whether patient allocation was concealed [Five studies -23 with oncealed  was remoP = 0.98). Self-management strategies decreased the risk of readmission due to heart failure . A funnel plot showed no evidence of publication bias.Three studies -20 with P = 0.76). There was no significant reduction in mortality with self-management   declare that they have no competing interests.The idea for the article arose from the discussion of AJ and SS, with input from JHL. AJ and an experienced research assistant conducted the literature search, in consultation with SS. AJ and SS reviewed all abstracts. All authors abstracted data and assessed the quality of the articles. AJ completed the statistical analysis of the results and prepared the draft of the submission. All authors collaborated on reviewing and finalizing of the paper. SS and JHL controlled the decision to publish. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Glomerulocystic kidney disease is an uncommon type of cystic renal disease. It is characterized by cortical microsysts, which are represented by cystic dilatation of Bowman's spaces.We describe a case of glomerulocystic disease in a neonate and another in an abortus associated with tracheo-oesophageal fistula and megacystic-megaureter syndrome. The kidney on autopsy was sponge-like and revealed presence of cysts corresponding to dilatations of Bowman's space microscopically. In these two cases, the Glomerulocystic Kidney Disease in one case corresponded to a sporadic form and, in the other, to a syndromic, non-heritable form of glomerulocystic kidney disease.The associated anomalies in Glomerulocystic Kidney disease are well described in the literature. Two more new unrelated associations are described in this article. Glomerulocystic kidney disease (GCKD) was characterized as a distinct histopathological entity in 1976 by Taxy and Filmer, who described this as a heterogeneous group of disorders with a common link being a variable degree of cystic dilatation of Bowman's spaces . FamiliaAbout one half of the examples of GCKD described in infants appear to be an expression of ADPKD with unusually early clinical onset, but not all cases of infantile-onset ADPKD have predominantly glomerular cysts. The exact significance of this morphologic expression in relation to ADPKD is yet not established. GCKD is also known to be encountered as sporadic condition in infants and young children who present in renal failure .th percentile of growth development according to crown-heal length. Externally, no congenital abnormalities were noted. The attached umbilical cord measured 10 cm and on transverse section showed presence of three vessels. Both the kidneys were normal sized with maintained reniform shape and a sponge-like appearance due to numerous, round, cystic spaces, which measured up to 2 mm . The second group comprises of diffusely glomerulocystic kidneys in newborns and young children, many of whom have family histories positive for classical polycystic kidney disease. It also includes sporadic and familial disease in older children and adults. No differences between familial and sporadic cases have been identified, apart from the family histories. The sporadic cases conceivably represent new mutations of the same disease. It also includes glomerulocystic kidneys as major component of heritable syndromes such as tuberous sclerosis, trisomy 13 and others. The third group includes glomerulocystic kidneys as components of diffuse cystic dysplasia and renal-hepatic-pancreatic dysplasia syndrome. The fourth group comprises all the sporadically occurring GCKD. Acquired GCKD group 5) has been described following hemolytic-uremic syndrome [ has beenThe mechanism of cyst formation in GCKD is poorly understood. Various factors that may be involved in the pathogenesis of GCKD include tubular obstruction due to renal medullary inflammation in neonates, increased intraglomerular pressure when the fetal glomeruli become functional and maternal intake of drugs such as phenacetin during pregnancy . Baxter Dilatation of the bladder and ureters can be seen with any obstructive lesion of lower urinary tract, but in the megacystic-megaureter syndrome, no anatomic obstruction is present . Severe The above two cases illustrates the sporadic form of GCKD (group 4) occurring in term baby and syndromic, non-heritable GCKD (group 3) in a 17-week abortus according to the above classification. A recent literature review failed to show a link between this rare association of GCKD with TEF, and megacystic-megaureter syndrome. Glomerular cysts are considered to be the basic and predominant lesions of GCKD. No dilatation of tubules is present; the absence of tubular dilatation excludes the diagnosis of ADPKD. Dysplastic elements were noted in the kidney sections of abortus, however were absent in term baby.In conclusion, the above two cases highlight the rare associations with which this heterogeneous entity is associated.


Adenocarcinoma of the oesophagus and gastro-oesophageal junction are rapidly increasing in incidence and have a well described sequence of carcinogenesis: the Barrett's metaplasia-dysplasia-adenocarcinoma sequence. During recent years there have been changes in the knowledge surrounding disease progression, cancer management and histopathology specimen reporting. Tumours around the gastro-oesophageal junction (GOJ) pose several specific challenges. Numerous difficulties arise when the existing TNM staging systems for gastric and oesophageal cancers are applied to GOJ tumours. The issues facing the current TNM staging and GOJ tumour classification systems are reviewed in this article. Recent evidence regarding the importance of several histopathologically derived prognostic factors, such as circumferential resection margin status and lymph node metastases, have implications for specimen reporting. With the rising use of multimodal treatments for oesophageal cancer it is important that the response of the tumour to this therapy is carefully documented pathologically. In addition, several controversial and novel areas such as endoscopic mucosal resection, lymph node micrometastases and the sentinel node concept are being studied. We aim to review these aspects, with special relevance to oesophageal and gastro-oesophageal cancer specimen reporting, to update the surgical oncologist with an interest in upper gastrointestinal cancer. In the Western world, distal oesophageal and gastro-oesophageal adenocarcinoma is increasing in incidence faster than any other type of gastrointestinal cancer . During There are many studies indicating that hospitals which manage large numbers of patients with upper gastrointestinal cancer have better outcomes -4, althoThe work load of the specialist gastrointestinal pathologist is increasing, especially in the assessment of oesophageal resection specimens and endoscopic biopsy reporting. There are many reasons for this increase:• The numbers of patients participating in Barrett's oesophagus surveillance programmes is increasing. A particularly controversial area is the designation of high grade dysplasia (HGD).• Tumours around the gastro-oesophageal junction, which are rapidly increasing in incidence, pose several specific problems to the histopathologist.• As with other tumour sites, standardisation and an emphasis on quality and completeness of pathological reporting have become mandatory. There is a need for pathologists to comply with the criteria in the minimum dataset for reporting oesophageal cancer specimens.• In some units the pathologist has a role in the assessment of the resected specimen immediately after surgical excision.• Newer evidence on the importance of several histopathological prognostic factors, including circumferential resection margin status and lymph node metastases, will have special implications for specimen reporting.• With the rising rates of multimodal treatments for oesophageal cancer the pathologist will have an increasing role in documenting the response of the tumour to this therapy.• The issues of endoscopic mucosal resection (EMR), lymph node micrometastases and the sentinel node concept have the potential to expand the scope of the histopathologist.We aim to review these aspects, with special relevance to oesophageal and gastro-oesophageal cancer specimen reporting, to update the surgical oncologist with an interest in upper gastrointestinal cancer.Accurate assessment of endoscopic biopsy material is crucial in the assessment of patients with Barrett's oesophagus, oesophageal epithelial dysplasia and adenocarcinoma. The British Society of Gastroenterology (BSG) has recently published guidelines for the diagnosis and management of Barrett's columnar-lined oesophagus . More biin-situ to try to increase inter-observer agreement, however in the UK dysplasia is still in use. Changes in the epithelial cells include lack of maturation towards the surface, variation in nuclear size and shape, nucleolar enlargement, increased cytoplasmic ratio, hyperchromasia and presence of abnormal mitoses. Architectural changes seen in dysplasia include stratification of nuclei with loss of the normal basal location. Barrett's dysplasia is classified into either indefinite for dysplasia, low grade dysplasia (LGD) or HGD by the degree of cellular and architectural changes [Dysplasia is defined as unequivocal neoplastic transformation of the epithelium, strictly confined within the basement membrane of the gland from which it arises . There i changes ,14. A di changes . FurtherAs a consequence of the subtle cytological changes from LGD to HGD, previous studies have shown that there are marked intra-observer and inter-observer variations in the classification of the degree of dysplasia . The cliAppropriate care of patients with HGD in the setting of Barrett's oesophagus relies heavily on the accuracy of reporting the degree of dysplasia . Early sTreatment options for HGD are controversial and range from intense endoscopic surveillance, endoscopic ablative therapy, EMR and oesophagectomy . In thosThe histopathologist has an important role in ensuring quality and a consistent approach to pathological reporting. An accurate histopathology report is essential for providing detailed staging of the primary tumour, elucidating prognostic information and for guiding optimal clinical management decisions. Audit, research, cancer registry data and other epidemiological studies rely heavily on the accuracy of this information. Upper gastrointestinal surgeons and clinicians have demanded an increase in the quality and quantity of information from the pathologist.The Royal College of Pathologists have published guidelines on the standards of oesophageal resection specimen reporting which include a proforma detailing the Minimum Dataset requirements . A similTumours around the gastro-oesophageal junction (GOJ) have become commoner in recent decades ,30,31 anGuidelines for the management of oesophageal and gastric cancer as 'it is uniform, allows data comparison from different centres, and is important for the stratification of patients in prospective studies' [The UICC/AJCC classification systems rely on the anatomical location of the epicentre or predominant mass of the tumour to decide whether the tumour is oesophageal or gastric in origin . With thstudies' . Criticistudies' -38.The existing TNM staging system also has some deficits , which i• It is based primarily on data from patients with squamous cell carcinoma of the middle and upper oesophagus• There is confusion regarding whether the oesophageal or gastric TNM systems should be used for GOJ tumours• Lymph node involvement beyond the regional lymph nodes is considered metastatic disease (M1)• The number of positive lymph nodes has been shown to be a strong prognostic factor by many authors, however, this is not apart of the current TNM systemCurrently there is confusion about whether the gastric or oesophageal staging systems should be used for the histological reporting of gastro-oesophageal junction tumours. Due to this confusion a tumour at the GOJ could be classified as oesophageal by the surgeon but gastric by the histopathologist or vice versa, especially when cases are not discussed at a preoperative multidisciplinary meeting. Usually, Type I adenocarcinomas are staged as oesophageal cancer and Type III as gastric cancer. Type II adenocarcinomas are staged as oesophageal cancer by some authors and as gastric by others. This is not ideal as there are significant differences between the gastric and oesophageal TNM staging systems in all three TNM categories Table . There aThe most important predictors of prognosis appear to be the overall TNM stage , completThe residual tumour classification Table  is one oThe circumferential resection margin (CRM) is defined as the surgically cut surface of the connective tissues that encase the oesophagus. Presence of tumour within 1 mm of this resection margin is classified as evidence of involvement. Although it has long been established that involvement of the proximal or distal resection margin is a poor prognostic factor ,47, the et al [et al., studied 135 patients who underwent oesophagogastrectomy [An initial study by Sagar et al  in 1993 trectomy , they intrectomy .In a follow-up study with larger patient numbers carried out by the same group , CRM waset al., [et al., had the lowest rate of CRM involvement (20%). Therefore the prognostic impact of CRM status may be related to the completeness of the mediastinal dissection. A similar situation is present in rectal cancer surgery, where the prognostic effect of CRM involvement is lessened following more radical surgical resection [Not all studies have shown a positive CRM to predict a poor prognosis in oesophageal cancer. Khan et al.,  observedet al., , suggestesection .et al., questioned whether CRM status should be an essential part of oesophageal resection specimen reporting [Although Khan eporting , the majeporting -53,56. Meporting ,52,56. IIt is important that the anatomy of the oesophagus is understood, especially as regards to the surrounding structures in the thorax. The CRM includes the whole circumference at and just above the gastro-oesophageal junction, but more proximally it is concentrated anteriorly and posteriorly with pleura on the lateral aspects. The pleural surfaces are not a true CRM and the significance of tumour involvement at this site is uncertain as there have been no large studies examining this . It can en-bloc. In addition, there is no specific fascial boundary equivalent to the mesorectal or Denonvillier's fascia present in the pelvis. CRM involvement in the oesophageal cancer specimen may therefore be much more a consequence of advanced tumour stage rather than the skill of the surgeon in carrying out a complete resection. Nevertheless, involvement of the CRM may be an indicator of the quality of pre-operative staging. At present most pathologists open oesophagogastric resections longitudinally, with or without pinning out of the fresh specimen, for fixation [In rectal surgery, a large intact capsule of mesorectum surrounding the resected cancer specimen has been suggested as a marker of good quality surgery ,59. Howefixation . This doSerosal invasion is an important prognostic marker in many gastrointestinal malignancies . Gastro-Vascular invasion is known to be a strong prognostic factor and is included in the oesophageal minimum dataset. However, it is not included in the gastric minimum dataset , which fThe presence of lymph node metastases is often the most significant independent factor on multivariate analysis ,43,64,65The current (2002) UICC guidelines recommend a minimum examination of 6 lymph nodes to classify a patient N0 . HoweverIn addition to a pure number based system, nodal involvement in relation to the lymph node capsule (intra or extracapsular) has recently been shown to be strongly prognostic on multivariate survival analysis in oesophagogastric adenocarcinoma ,72. The Immunohistochemical techniques can identify micrometastases which are missed by standard haematoxylin and eosin staining. Cytokeratin, a component of the cytoskeleton of epithelial cells, is not found in normal nodes enabling monoclonal antibodies to certain cytokeratin markers (such as AE1/AE3) to be used to detect micrometastases. These techniques may detect single tumour cells or cell clusters in lymph nodes that have been staged as tumour free on routine examination. The prognostic outcome of the detection of micrometastases detected by immunohistochemistry is controversial as some studies have found an association with increased risk of tumour recurrence and decreased survival -75, but The importance and clinical utility of the sentinel node concept has been extensively evaluated in malignant melanoma and breast cancer. It is being evaluated in gastrointestinal cancers , includiInitial feasibility studies have been performed in oesophageal cancer ,80. The Endoscopic mucosal resection (EMR) techniques are being increasingly used for treatment and staging of superficial early cancers of the oesophagus, especially in Japan although Western centres are gaining experience ,82. AlthMultimodal therapy, in particular neo-adjuvant treatment prior to surgical resection, is increasingly used in oesophageal cancer. The aims of neo-adjuvant chemotherapy, with or without radiotherapy, are to downstage or 'sterilise' the primary to improve the likelihood of complete tumour (R0) resection, reduce tumour recurrence, treat occult micro-metastases and ultimately to improve overall survival. Although there is some evidence from randomised controlled trials that neo-adjuvant therapy in addition to surgery can prolong survival -88, therA pathological complete response (CR) occurs in less than 30% of patients who undergo surgery after preoperative chemotherapy or chemo-radiotherapy -94. AlthIn instances of a potential CR, knowledge of the precise location of the tumour before neo-adjuvant therapy is crucial to direct pathological sampling as the oesophagus may look normal macroscopically. However, the precise number of tumour blocks to take has yet to be clarified and currently is at the discretion of the histopathologist. Various architectural, nuclear and cytoplasmic changes in tumour and non-tumour tissue have been described following neo-adjuvant chemotherapy . In addiet al., system [There are a variety of different grading systems for measuring residual tumour in oesophageal resection specimens after preoperative chemotherapy, with or without radiotherapy Table  101-103103. The , system , whereas, system . It alsoWith the increased interest in multi-modal therapy, however, the classification of pathological response is of increasing importance to the histopathologist and surgeon. Proposals to revise the oesophageal cancer staging system to accommodate the pathological response of the tumour following preoperative chemo-radiotherapy have recently been made . The finThere is great interest in evaluating predictive factors for patient response to neo-adjuvant chemo-radiotherapy. An accurate predictive factor would allow the targeting of therapy to patients who are most likely to achieve a benefit, while those that are unlikely to respond can avoid potentially toxic therapy and receive earlier surgery.A recent study has shown that patients with signet-ring cell or mucinous histology on pre-treatment biopsies have an improved response and better overall survival when treated with neo-adjuvant chemo-radiotherapy . This stMore sophisticated molecular techniques have been evaluated on pre-therapeutic biopsies in an attempt to find a good predictive marker. Although, none of these are currently being used in routine clinical practice, the role of the histopathologist in this regard is likely to increase in the future.The management of patients with oesophagogastric cancer should be discussed at all key points in the patient journey at specialist multidisciplinary team (MDT) meetings . There iAccurate assessment of endoscopic biopsy material is crucial in the assessment of patients with Barrett's oesophagus. As appropriate care of patients with HGD in the setting of Barrett's oesophagus relies heavily on the accuracy of reporting the degree of dysplasia, standardised methods and guidelines should be followed.The classification and staging systems for GOJ tumours need to be improved and future research into this area is greatly warranted. A better understanding of the clinical relevance of each classification system for GOJ tumours needs to be achieved before a final recommendation is made. Although the Siewert classification has been shown to have some clinical relevance, other authors have found that it unnecessarily complicates the assessment of these tumours and is fraught with difficulties because of the overlapping nature of tumours in this region. Some authors would argue that instead of three staging systems, only two are required . Further clinical studies to address these issues are urgently required.There is already sufficient evidence to confirm that CRM involvement is a marker of poor prognosis in oesophageal cancer. As such, CRM status must continue to be routinely reported. However, our understanding of its full significance is limited compared to the field of rectal cancer where CRM has been extensively studied. For example, studies have been carried out directly comparing histopathological sections with preoperative cross-sectional imaging. In rectal cancer, this information has been used to predict potential CRM involvement prior to surgery and thus the need for neo-adjuvant therapy. In future oesophageal studies, especially prospective trials involving neo-adjuvant therapy or the comparison of different surgical techniques, it is imperative that involvement of the CRM is analysed.There are exciting new research opportunities in the identification of lymph node micrometastases and sentinel node involvement; however they have yet to be proven clinically useful. There is emerging evidence that the histopathological evaluation of the tumour response to neo-adjuvant therapy is prognostically relevant. However, further research studies are required to confirm its role in patient management. Although several classification systems have been devised they have yet to be agreed for routine clinical use. A standardised classification system for the assessment of residual tumour burden after neo-adjuvant therapy will need to be agreed and ideally adopted internationally.The author(s) declare that they have no competing interests.EG conceived of the original manuscript idea, performed the literature search and wrote the first draft. SP, NM and IW revised subsequent versions of the manuscript for intellectual content. All authors read and approved the final manuscript.


Clinical utilization of ovulation stimulation to facilitate the ability of a couple to conceive has not only provided a valuable therapeutic approach, but has also yielded extensive information on the physiology of ovarian follicular recruitment, endometrial receptivity and early embryo competency. One of the consequences of the use of fertility enhancing agents for ovarian stimulation has been the creation of a hyperestrogenic state, which may influence each of these parameters. Use of aromatase inhibitors reduces hyperestrogenism inevitably attained during ovarian stimulation. In addition, the adjunct use of aromatase inhibitors during ovarian stimulation reduces amount of gonadotropins required for optimum stimulation. The unique approach of reducing hyperestrogenism, as well as lowering amount of gonadotropins without affecting the number of mature ovarian follicles is an exciting strategy that could result in improvement in the treatment outcome by ameliorating the deleterious effects of the ovarian stimulation on follicular development, endometrial receptivity, as well as oocyte and embryo quality. Current epidemiological evidence suggests that 15% of couples will experience infertility. Background prevalence rates now appear to be reasonably stable, but there is evidence of an increase in the rate of referrals for medical help . Farley After correcting the abnormalities detected during the diagnostic workup, ovulation induction is usually performed either for treatment of anovulation/oligo-ovulation, or empirically in regularly ovulating women. This approach results in a pregnancy rate of around 8%–15% per cycle depending on the agents used for ovulation induction and the characteristics of the couple, such as the woman's age and the presence or absence of a male factor. Couples who do not become pregnant with ovulation induction alone then undergo more sophisticated treatment modalities including intrauterine insemination (IUI) and in-vitro fertilization and embryo transfer (IVF-ET) as a treatment of last resort , fertilization, and embryo culture techniques have led to an optimization in the number and quality of embryos available for ET. In contrast, uterine receptivity has failed to benefit from parallel improvements, and its disarrangement is likely to represent an important cause of the sub-optimal embryo implantation rates observed in IVF-ET .In the following section we review in brief both animal and human evidence for the unfavorable outcome including impaired implantation and increased adverse outcomes in pregnancies achieved following ovarian stimulation when compared with spontaneous pregnancies.Increased pre- and post-implantation embryonic loss has been reported in mammals -21 incluThe poor outcome after ovarian stimulation has been attributed to adverse effects on the maternal side and or the gametes and embryo side. On the maternal side: inadequate uterine synchrony or receptivity has been reported. On the gamete and embryo side, ovarian stimulation has been found associated with chromosomal defects in the oocyte leading to increased lethality during the preimplantation stages ,23. HoweMany studies have found higher pregnancy rates in donor oocyte recipients than patients undergoing standard IVF-ET ,25. The Another evidence for adverse effects of ovarian stimulation on implantation is the higher implantation rate associated with IVF-ET in the natural or un-stimulated cycle. Although most studies were associated with a rather high proportion of cancelled cycles [25–75%] and a low clinical pregnancy rate per started cycle [range 0–23%], higher implantation rates have been reported (up to 30%) .Induction of ovulation has been shown to raise the risk of miscarriage when compared with spontaneous pregnancies . This waWhile ovarian stimulation has been suggested to contribute, at least in part, for the adverse obstetric outcome other studies have shown that infertility itself is a factor that leads to increased obstetric risks and that sub-fertility is a predictor for low birth weight. Some believe that it is the cause of infertility itself, rather than the use of ovarian stimulation, that is the reason behind poor obstetric outcome after infertility treatment ,39.Several causes and targets have been suggested to explain the poor outcome associated with ovarian stimulation Table . These iIt is believed that the supraphysiological levels of estrogen, attained during ovarian stimulation, may explain, at least in part, for the adverse effects of ovarian stimulation on the outcome of infertility treatment -42. Diff2) concentrations were > 2500 pg/ml [>9000 pmol/L.] compared with patients having lower E2 concentrations. High serum E2 concentrations on the day of hCG injection in high and normal responder patients, regardless of the number of oocytes retrieved and the serum progesterone concentration were found to be detrimental to uterine receptivity   variable in assessing endometrial receptivity seems to be limited . One of Histological studies have confirmed that the sub-endometrial halo surrounding the endometrium represents the innermost layer of the myometrium, and compared with the outer myometrium, it consists of a distinct compartment of more tightly packed muscle cells with increased vascularity. Studies have shown that interactions between the junctional zone and the endometrium may play an important role in the implantation process ,90 and eMany investigators have also noted the correlation of junctional zone contractions with pregnancy outcome in both natural  and assiKupesic et al. compared the 2-D and 3-D ultrasonographic scoring systems by combining parameters including endometrial thickness, volume, echogenicity, and sub-endometrial blood flow. They found the two systems had similar efficiencies in predicting pregnancy outcome of IVF-ET procedures . Several2 administration improves endometrial proliferation and uterine perfusion, presumably because of combined local and systemic effects, but may interfere with P-induced uterine relaxation -methionine uptake  surge, luteal phase inadequacy was reported. A meta-analysis of different clinical trials demonstrated beneficial effects of luteal support when ovarian stimulation was carried out with human menopausal gonadotropins [hMG] in association with GnRHa .Recently, GnRH antagonists have become available for clinical use. Whether GnRH antagonists induce down-regulation of pituitary GnRH receptors is still a subject of investigation . It has 2/P) ratio was considered a main cause of luteal-phase inadequacy, possibly through the luteolytic action of E2  surge during ovulation and continuous tonic LH pulses during the luteal phase are necessary for the proper development of the corpus luteum. Corpus luteum dysfunction due to the effect of ovarian stimulation on the pituitary gland and hypothalamus will be discussed later.Normal corpus luteum function requires optimal follicular development in the follicular phase, especially follicle stimulating hormone (FSH) stimulation, adequate luteinizing hormone (LH) surge during ovulation and continuous tonic LH pulses during the luteal phase. In turn, the normal luteal phase is characterized by an optimal hormonal environment and adequate endometrial secretory transformation. As many factors contribute to a normal corpus luteum function, any alteration might exert a deleterious effect on the final target, the endometrium, leading to embryo/endometrial asynchrony . Luteal The lifespan and steroidogenic capacity of the human corpus luteum is dependent on continuous tonic luteinizing hormone secretion as well as healthy adequate gonadotropins surge. Feedback mechanisms from ovarian steroids and GnRH pulses regulate LH secretion during the luteal phase, but a number of autocrine and paracrine factors within the ovary might also play a role in controlling corpus luteum function .It is obvious that during COH for ART, the prevention of endogenous LH surge is mandatory to avoid the occurrence of premature LH surge as well as for timing of oocyte retrieval.However, during ovarian stimulation cycles in which no pituitary suppression is used, there are data that suggest a defective endogenous gonadotropins surge. The gonadotropins surge is an event crucial for final oocyte maturation, ovulation, and subsequently for corpus luteum function. The duration of the LH peak seems to be more important than its amplitude for the induction of ovulation . Ovulati2 levels, the number of follicles, and the total follicular fluid volume aspirated at laparoscopy. This suggests that during superovulation induction for IVF-ET, the endogenous LH surge is attenuated by factors, which are related to the degree of ovarian hyperstimulation , [Higher follicular fluid leptin concentrations correlated with lower intrafollicular oxygen concentration [pO2],  a condit2],  with a d2],  who foun2], . Moreove2],  a role f2], .2 has been reported to increase leptin mRNA expression. In human adipose tissue culture, E2 stimulated leptin secretion in women but not in men  are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from IVF-ET. During the last few years, concerted efforts have focused on reducing these rates by restricting the number of embryos to transfer .In order to fulfill this task, several approaches have been suggested to improve the outcome of treatment after ovarian stimulation and assisted reproduction. Table We suggest a THIRD novel approach that we believe it may carry a hope for improving the outcome of treatment after ovarian stimulation and assisted reproduction. This approach involves the use of aromatase inhibitors.Different approaches have been suggested to improve the treatment outcome during assisted reproduction by reducing the intensity of ovarian stimulation that would reduce the high estrogen levels . These aHowever, all these measures are associated with the major drawback of affecting the main goal of ovarian stimulation, which is the achievement of a suitable number of embryos when a good number of oocytes retrieved.The first IVF-ET treatment in the literature was a natural cycle IVF-ET. Since then natural cycle IVF-ET has been largely replaced by IVF-ET with ovarian stimulation. Natural cycle IVF-ET has several advantages including a close to zero multiple pregnancy rate, and a zero risk of ovarian hyperstimulation syndrome as well as being less time consuming, physically and emotionally less demanding for patients, and cheaper than stimulated IVF-ET. However, it seems to be less effective. Unfortunately, good quality randomized controlled trials and formal cost-effectiveness analyses are lacking. Pelinck reviewed 20 selected studies that comprised a total of 1800 cycles of natural cycle IVF-ET, resulting in 819 embryo transfers (45.5% per cycle) and 129 ongoing pregnancies (7.2% per cycle and 15.8% per embryo transfer). The authors concluded that: in spite of being a low-risk, low-cost and patient-friendly procedure, high cancellation rates because of premature LH rise and premature ovulations hamper efficacy of natural cycle IVF-ET and that a randomized controlled trial comparing natural cycle IVF-ET with current standard treatment strategies is warranted. .The cumulative live birth rates after four cycles of treatment was reported to be 32% which is comparable with the value of 34% for women having conventional IVF-ET treatment . HoweverIn the implantation rate per embryo after natural cycle IVF-ET was found quite acceptable up to 50%. Also, the fertilization rate was found to be 100% for the oocytes retrieved from the mature follicles (as reviewed by  Pelinck)Minimal stimulation for IVF-ET that is less expensive than full stimulation and minimizes monitoring and patient discomfort has been described for almost 10 years and found to be associated with acceptable pregnancy rates and was suggested as an attractive alternative to select patients undergoing IVF-ET . This pr2 levels during the preimplantation period , and already approved for clinical use to reduce estrogen production in postmenopausal women with breast cancer.They have not been used in women of the reproductive age group. However, we have found these aromatase inhibitors to be effective in inhibiting the aromatase enzyme and effectively decrease estrogen levels in women of the reproductive age group during their successful use for ovarian stimulation ,431,432.2 level/mature follicle) when compared with conventional stimulation protocols . Such low E2 levels may be beneficial and explains at least partially the improved outcome of treatment in terms of achieving promising high pregnancy rates during aromatase inhibitor treatment.In our experience with the use of an aromatase inhibitor for ovarian stimulation, estrogen levels were significantly lower  or by down-regulation of the GnRH receptors at the pituitary levels resulting in pituitary desensitization and prevention of the release of endogenous LH ,455.Unfortunately, the use of GnRH analogues , is associated with several problems including increasing the dose of FSH required to achieve optimum COH , as well as the luteal phase defect due to a dysfunctional corpus luteum function secondary to persistent endogenous LH suppression as explained earlier. In addition, there is rising evidence of a possible direct deleterious effect of the GnRH analogues, especially the antagonist at the level of the endometrium .The use of an aromatase inhibitor to reduce estrogen levels attained during COH may be effective in preventing the occurrence of premature ovarian surge. This would avoid the use of GnRH analogues during stimulation protocols for assisted reproduction which has several advantages including prevention of the possible deleterious effects of these agents as mentioned above in addition to reducing the cost of treatment as well allowing implementing much simpler stimulation protocols during assisted reproduction.The expression of aromatase enzyme in endometriotic tissues with the significant role of locally produced estrogen in endometriosis progression  suggestsThere are three main concerns that may arise regarding the use of the aromatase inhibitors to improve the outcome of treatment after ovarian stimulation and assisted reproduction. They include the possible deleterious effect of the low follicular estrogen milieu on the development of the oocytes, the possible direct effect of the aromatase inhibitors on the oocyte development, fertilization or embryogenesis, and the accumulation of the androgens that may result from inhibition of their conversion into estrogens.17-hydroxylase/17–20 lyase  and their size distribution (as assessed by ultrasonography) proved comparable for control and ATD-treated animals. Similarly, no difference was noted in the number of oocytes collected or in the proportion of oocytes reinitiating meiosis (MI at the time of collection). In contrast, ATD-treated animals displayed a marked increase (31 vs. 11%) in the proportion of prophase I oocytes. Moreover, ATD-treated oocytes displayed retarded in vivo completion of maturation to MII (4% vs. 26%). Interestingly, the latter retardation was not observed in vitro. Furthermore, two of the four ATD-treated animals yielded oocytes that were morphologically abnormal. Finally, oocytes from ATD-treated animals displayed significantly reduced rates of fertilization (9% vs. 25%) as compared with controls. However, the cleavage rate after successful fertilization was similar for ATD-treated vs. ATD-untreated controls. In vitro cultures of granulosa cells collected at the time of oocyte aspiration revealed equivalent 24-h progesterone production in treated and control animals. The authors concluded that these findings suggest that the acute reduction in E2 levels during the terminal stage of gonadotropin stimulation had little effect upon follicular recruitment and expansion but an apparent detrimental effect upon gametogenic function may, in fact, exist. However, we cannot extrapolate from this study that the findings are simply due to the acute reduction of estrogen production as it is important to realize that there was a concomitant significant rise in androstenedione. Moreover, the time of administering an aromatase inhibitor during the latter part of the follicular phase as well as the irreversible nature of aromatase inhibition constitute important differences from our model of using a REVERSIBLE aromatase inhibitor, TEMPORARILY, EARLY in the menstrual cycle that has a SHORT half-life.In cycling female rhesus monkeys, the aromatase inhibitor, 1,4,6-androstatrien-3, 17-dione (ATD) was used to inhibit estrogen production during gonadotropin ovarian stimulation . Animals2, respectively without obvious effect on follicular maturation, ovulation, and luteal function as assessed by serum hormone profiles as well as by laparotomy. Moreover, the concurrent administration of FSH and an aromatase inhibitor resulted in the suppression of the FSH-induced increase in the circulating levels of E2 (by 100%) with no effect noted on either the number of follicles developed or their size relative to control. In addition, granulosa and theca cells, removed on day 9 of the treatment cycle, were responsive to gonadotropins in vitro, disclosing no evidence of a deleterious effect on the cellular development and maturation of follicular cells.Selvaraj ,481 and 2 levels in mediating the adverse effects of hyperstimulation with PMSG on early embryonic development in the rat [2 production. The authors conducted three experiments. In the first, varying doses of 4-OHA were administered either concurrently with hCG to pro-estrus female rats hyperstimulated at early diestrus stage with 20 IU PMSG or alone into non-hyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 mg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females with optimum protection at 2000 mg, while low doses of 100 and 500 mg/rat were ineffective. When administered alone, only the highest dose of 5000 mg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 mg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and high doses of 4-OHA (5000 mg/rat) was needed to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 mg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. These results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. The authors concluded that this might be due to the suppression of estrogen production, thereby alleviating the supraphysiological level of E2, which is typically present in PMSG-treated females, which supports the hypothesis that excessive E2 is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.Terry studied a possible role of high E the rat . They usThe high success rates of ovulation and achievement of pregnancy in our reports on the use of aromatase inhibitor in ovarian stimulation, despite significantly lower estrogen levels could be due to several reasons. First: We used one of the third-generation "REVERSIBLE" aromatase inhibitors. Second: The aromatase inhibitor was administered EARLY in the follicular phase and for a limited period of time, which would allow the rapid clearance of the aromatase inhibitor from the body due to its SHORT half-life. Third: the reversible nature of the aromatase enzyme inhibition and the rising levels of FSH, which induce the expression of the aromatase enzyme, both would not allow for the estrogen levels to drop drastically below the low physiologic range. We believe that these low estrogen levels attained during the use of aromatase inhibitors for ovarian stimulation are compatible with healthy development of the oocytes, fertilization, embryo development, implantation and achievement of pregnancy. Fourth: the absence of significant rise in androgen levels would have prevented any possible unwanted effects on the terminal part of the oocyte maturation and ovulation.As explained above, the short half-life of the aromatase inhibitors and limiting the administration to the early part of the follicular phase would allow the rapid clearance of the medications from the body before the important stage of fertilization and embryogenesis. This in addition to the absence of accumulation of the aromatase inhibitors of any of their metabolites would make them safe for the ovarian stimulation. We have reported a preliminary data on the pregnancy outcome after the use of aromatase inhibitors for ovarian stimulation supporting the safety of using these medications for such indication .Data have been accumulating regarding the absence of deleterious effects in association with aromatase inhibitor treatment on follicle/oocyte maturation and embryo development in mice ,493. TreAs mentioned before, the rapid clearance of the aromatase inhibitors from the body due to their short half-life and the reversible nature of the aromatase enzyme inhibition as well as the rising levels of FSH, which induce the expression of the aromatase enzyme, all would not allow the clearance of any accumulating androgens by converting them to estrogen. The conversion of the androgens into estrogens  is a terminal step in the cascade of steroidogenesis. So, substrate (androgens) accumulation is not expected to be very significant, as other alternative earlier steps in the steroidogenesis pathways will work to clear out the accumulating androgens. For these reasons, pharmacokinetic studies on the new aromatase inhibitors reported the absence of significant androgen elevations or abnormal changes in other steroids in patients receiving aromatase inhibitors for breast cancer. In a subset of our patients who received an aromatase inhibitor for ovarian stimulation, we did not find significant change in the androgen levels while receiving the medication when compared to stimulation with gonadotropins alone (unpublished data).Most recently we published data on the favorable pregnancy outcome after the use of letrozole for ovarian stimulation alone or in conjunction with gonadotropins . This stOvarian stimulation particularly in conjunction with assisted reproductive technologies aims at stimulating the recruitment of several mature ovarian follicles that would enhance the chance of successful treatment by obtaining several embryos readily available for transfer into the uterus. It is obvious that supraphysiological levels of estrogen are inevitably attained during ovarian stimulation due to the significant contribution of estrogen production from each one of the several mature ovarian follicles. There is growing evidence that such supraphysiological estrogen levels are deleterious on the development of the endometrium, oocytes, embryos as well as other targets through different mechanisms. This is believed to explain, at least in part, the low pregnancy rates associated with assisted reproduction, mainly as a result of the persistently low implantation rates that have not improved impressively along almost three decades of assisted reproduction experience. This led several investigators to adopt the concept of minimal ovarian stimulation for assisted reproduction as an alternative approach to reduce the deleterious effects of the supraphysiological estrogen levels attained during routine controlled ovarian hyperstimulation. Unfortunately such approach, despite being logical, is associated with the major drawback of achieving fewer oocytes than desired, which, expectedly, reduces the chances of treatment success. Recently, we reported the success of the new generation aromatase inhibitors in ovarian stimulation. We found these agents to be effective in reducing the amount of estrogen production by mature ovarian follicles significantly. Furthermore, we found the use of these agents to be associated with improved ovarian response to stimulation by gonadotropins, resulting in significant reduction in the dose of gonadotropins required for controlled ovarian hyperstimulation. The novel idea of using aromatase inhibitors during ovarian stimulation for assisted reproduction combines the benefit of a significant reduction in estrogen production as well as gonadotropins dose, with the exiting advantage of achieving a good number of mature oocytes as a result of enhanced ovarian response to gonadotropins. This is expected to improve the various aspects of treatment outcomes after assisted reproduction including increased safety, reduced cost, as well as enhanced implantation rate. If confirmed in well-designed clinical trials, this would facilitate acceptance of the concept of single embryo transfer by infertile couples and practitioners, thereby reducing the epidemic of multiple births after assisted reproduction with its significant deleterious health and economical effects. This is particularly true in the light of recent data supporting the success of the approach of single embryo transfer -497.MFMM has written the review article, RFC and MPD have reviewed the manuscript