Data about the entire sperm DNA methylome are limited to two sperm donors whereasstudies dealing with a greater number of subjects focused only on a few genes orwere based on low resolution arrays. This implies that information about what wecan consider as a normal sperm DNA methylome and whether it is stable amongdifferent normozoospermic individuals is still missing. The definition of theDNA methylation profile of normozoospermic men, the entity of inter-individualvariability and the epigenetic characterization of quality-fractioned spermsubpopulations in the same subject  are relevantfor a better understanding of pathological conditions. We addressed thesequestions by using the high resolution Infinium 450K methylation array andcompared normal sperm DNA methylomes against somatic and cancer cells. Ourstudy, based on the largest number of subjects (n = 8) everconsidered for such a large number of CpGs ,provided clear evidence for i) a highly conserved DNA methylation profile amongnormozoospermic subjects; ii) a stable sperm DNA methylation pattern indifferent quality-fractioned sperm populations of the same individual. Thelatter finding is particularly relevant if we consider that different qualityfractioned sperm subpopulations show differences in their structural features,metabolic and genomic profiles. We demonstrate, for the first time, that DNAmethylation in normozoospermic men remains highly uniform regardless the qualityof sperm subpopulations. In addition, our analysis provided both confirmatoryand novel data concerning the sperm DNA methylome, including its peculiarfeatures in respect to somatic and cancer cells. Our description about a highlypolarized sperm DNA methylation profile, the clearly distinct genomic andfunctional organization of hypo- versus hypermethylated loci as well as theassociation of histone-enriched hypomethylated loci with embryonic development,which we now extended also to hypomethylated piRNAs-linked genes, provides solidbasis for future basic and clinical research. The rationale behindselection is mainly related to a predicted higher functional competency and a highergenomic integrity of selected spermatozoa. Interestingly enough, despite the sametesticular environment, biochemical markers Apart from the large inter-individual variability of the above mentioned quantitativetraits of spermatogenesis, semen of normozoospermic men contains a qualitatively (interms of motility and morphology) heterogeneous sperm population. With the adventand diffusion of assisted reproductive techniques, a number of sperm selectionmethods have been developed in order to obtain sperm subpopulations enriched withhighly motile and morphologically normal spermatozoa to be used for de novo methylation) which may generate substantial epigeneticvariability. The sole study addressing the question about intra- andinter-individual DNA methylation changes in normozoospermic men was based on a12,198-feature CpG island microarray Given that epigenetic signals such as DNA methylation and histone modifications arecrucial for the proper functioning of the genome, phenotypic differences in spermproduction  at both inter- andintra-individual level may also be due to an epigenetic variation. This hypothesisseems to be plausible if we consider that the epigenome of mature spermatozoamirrors a series of sequential epigenetic reprogramming events . This peculiar class of small non coding RNAs arespecifically expressed in the testis and seem to be involved in the maintenance ofgenomic stability and germ cell function through the silencing, via DNA methylation,of mobile genetic elements such as transposons  wholesperm population after 1 hour from semen collection; 2) swim-up fraction; 3)swim-down fraction. For sample EC7, the swim-down fraction has been excluded dueto DNA degradation. For 3 samples whole, semen at 2 Sperm DNA methylationprofile largely hours (corresponding to the time at which the swim-up procedureends) were also available for the comparison with the other fractions.Whole semen has been centrifuged on a 25% Percoll gradient (20 minutes)before the standard swim-up separation technique. Although much care was takenfor the selection of samples in terms of lack of contaminating cells, thispreliminary step further ensured the purity of the sperm population. The swim-upprocedure allows spermatozoa with progressive motility to “swim up”into the culture medium while hypomotile/immotile spermatozoa remain behind. Theupper fraction is denominated “Up”, whereas the fraction containinghypo/immotile spermatozoa is indicated in this manuscript as “Down orDn”.Sperm DNA was extracted with an user-developed version of the QIAamp®DNeasy&Tissue Kit purification protocol. Fresh washed (in PBS) sperm wasincubated 1∶1 with a lysis buffer containing 20 mM TrisCl (pH 8), 20mMEDTA, 200 mM NaCl and 4% SDS, supplemented prior to use with 100 mM DTTand 250 ug/ml Proteinase K. Incubation was performed for 4 hours at 55°Cwith frequent vortexing. Prior to processing in the columns, 200 ul of absoluteethanol and 200 ul of the kit-provided lysis buffer were added to the samples.Then, purification was performed according to kit instructions.DNA was quantified by Quant-iT™ PicoGreen dsDNA Reagent (Invitrogen) andthe integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of600 ng of each sample was performed according to the manufacturer'srecommendation for Illumina Infinium Assay. Effective bisulphite conversion waschecked for three controls that were converted simultaneously with the samples.4 µl of bisulfite converted DNA were used to hybridize on InfiniumHumanMethylation 450 BeadChip, following Illumina Infinium HD Methylationprotocol. Chip analysis was performed using Illumina HiScan SQ fluorescentscanner. The intensities of the images are extracted using GenomeStudio (2010.3)Methylation module (1.8.5) software. Methylation score of each CpG isrepresented as beta (β) value . The 450K DNA Methylation arrayincludes 485,764 cytosine positions of the human genome. From these cytosinesites, 482,421 positions (99.3%) are CpG dinucleotides, whilst only 3,343sites (0.7%) correspond to CNG targets. Thus, from this point on we willuse the term CpG, except when we refer specifically to putative CNG methylation.A general depiction of the 450K platform design, regarding functional genomedistribution, CpG content and chromosome location, is reported in a previousvalidation study from our laboratory The 450K DNA methylation array by Illumina is an established, highly reproduciblemethod for DNA methylation detection and has been validated in two independentlaboratories beta value in the 450 K platform is accompanied by adetection p-value. We based filtering criteria on the basis of these p-valuesreported by the assay. We examined two aspects of filtering out probes andsamples based on the detection p-values, selecting i) a threshold and ii) acut-off. Previous analyses indicated that a threshold value of 0.01 allows aclear distinction to be made between reliable and unreliablebeta values Every In order to identify differentially methylated CpG sites between differentquality fractioned sperm populations, a non parametric test (Wilcoxon rank sumtest) has been performed. Linear regression coefficient has been calculated(Spearman's rho) both for intra and inter-individual variability ofmethylation levels. For all comparisons of methylation levels between differentsubgroups Fischer exact test was performed.ia and ibrepresent the beta value for the i-essim CpG of samples “a” and“b”, and “n” the number of CpG sites selected.For the estimation of the degree of epigenetic dissimilarity between individualswe measured the Euclidean distances between two samples using the followingequation:In addition, to estimate the inter-individual variability of the methylationstatus in the promoters of the 6 genes previously described as highly variable,we calculated for each gene a 100,000 permutations test with the distances ofthe three groups, in order to obtain a random distribution of possible meandistances and get a p-value for the mean variability among individuals in agroup (the area below the distribution curve).For the estimation of enrichment in biological processes we performed ahypergeometric test on biological processes defined by Gene Ontology The ejaculate of a normozoospermic man contains a qualitatively heterogeneoussperm population . This part of the analysis focused on intra-individualvariation and addressed the biological question whether there are significantdifferences in methylation profiling between the “up”  versus “down” semenfractions in each subject.2 = 0.9896 toR2 = 0.9982) between “good” and“poor” quality sperm suspensions in all subjects  showing DNA methylation changes ininfertile men compared to normozoospermic controls as well as in differentquality-fractioned sperm populations (such as DAZ andDAZL). In all cases, a homogeneous methylation patternwas observed in the two fractions derived from the same individual and,accordingly, the two sperm fractions derived from all analyzed subjects didnot cluster separately  between individuals; ii) we measured theepigenetic distance  between the methylation level of CpGs in differentsubjects. The number of differentially methylated loci i.e. showing a SD>0.2 between different subjects was 1,591 in the whole semen, 1,207 inthe “down” fraction and 1,675 in the “up” fraction.This implies that for all comparisons the number of CpGs above theestablished threshold level was very low, representing <0.3% ofall loci tested. The GO analysis of genes related to the 1,675differentially methylated sites did not show any germ cell specificfunction. (data not shown).In order to further explore inter-individual variability, we analyzed eachtype of sperm fraction using two additional approaches: i) we quantified thenumber of CpGs showing a standard deviation >0.2 in the methylation level in respect to the swim-up fraction . HoweverBRCA1, BRCA2, HTT (HD), DMPK (DM1),PSEN1, PSEN2 by Flanagan et al. HTT,DMPK, PSEN1,PSEN2) in the swim-down sperm fraction , while we observed no relevantvariation in the swim-up fraction. In the whole sperm population sample,significance was reached only for BRCA1 (0.01136).Significant DNA methylation variations have been reported for promoters ofthe following 6 genes: Given that the swim-up fraction, being enriched in the best qualityspermatozoa, is the one used for assisted reproductive techniques, we aimedto provide a detailed description of genome wide DNA methylation profile ofthese cells. The average DNA methylation level of the 485,317 CpG sites was45% . However, an interesting feature of thesperm DNA methylome is the polarization of DNA methylation level towards thetwo extremes: 86% of all markers are either severely hypomethylated(<20%) or strongly hypermethylated (>80%). Intermediatemethylation level (20–80%) was observed only for 14% ofCpGs. We defined, in each subject, the number of hypomethylated andhypermethylated loci for the whole genome as well as for the sex chromosomesand autosomes, separately . The coeThe separate analysis of autosomal , X-linked  and Y-linked CpGs  revealed thatX-linked loci are significantly more frequently hypomethylated thanautosomal loci , as wasthe case also for the Y–linked loci  . On the Subsequently, we identified loci displaying the same DNA methylation patternin all subjects (“conserved” hypo or hyper) as well as locishowing different DNA methylation patterns . We analyzed the functionalgenomic distribution , CpGcontent and neighborhood context. For the latter, we referred to: i)“island” as a DNA sequence (>200-bp window) with a GC contentgreater than 50% and an observed: expected CpG ratio of more than0.6.; ii) “shore” as a sequence 0–2 Kb distant from theCpG island; iii) “shelf” as a sequence 2–4 Kb distant fromthe CpG island; iv) “open sea/others” as the remaining sequence.The methylation categories were also analyzed in relationship with genomiclocations related to RNA transcription .Sharp differences were observed between the “conserved” hypo andhyper-methylated loci according to the functional genomic distribution aswell as for the CpG and neighborhood context . Among aDifferentially methylated loci  have been identified only for 0.3% of all analyzed CpGs.Interestingly, the percentage of “variable” regions were lowerin X-linked loci (0.2%) and were completely absent in the 416Y-linked loci. Intriguingly, the pattern of “variable” CpGs wasmore similar to the “conserved” hypermethylated loci than to the“conserved” hypomethylated ones, as a matter of fact thevariation in DNA methylation between individuals is more pronounced inCpG-poor regions such as gene body, intergenic and “open sea”.Our next question was whether hypo and hypermethylated loci were linked tospecific biological processes. By performing GO analysis, we found that thetwo methylation patterns are involved in completely distinct cellularprocesses . An outsIn a previous study, Hammoud et al.−4) and was more heterogeneous. An additionaldatum supporting the link between hypomethylation and histone retention ofdevelopmental genes was that among the 106 developmental genes available inthe array, 62 presented in their promoters CpGs with <20% ofmethylation level as well as histone retention. On the contrary, no overlapwith histones was observed in developmental gene promoters showinghypermethylation.In agreement with Hammoud et al. The average percentage of hypomethylated loci was significantly higher insperm cells of the 8 subjects at the global, autosomal and sex-chromosomallevels  compared to the differentiatedsomatic cell .On the http://pirnabank.ibab.ac.in/) with the positions of the487,517 CpGs available in the array. In order to include potentialregulatory sequences, we extended the piRNA positions to ±2 Kb andthrough these criteria a total of 2,591 unique piRNAs have been found to becovered by 7,528 CpGs on the array. In spermatozoa, 80% of the totalarray-available piRNA-linked CpGs (n = 6050) revealedeither very high or very low methylation levels. In fact, similarly to theglobal sperm DNA methylome, sperm piRNA-linked CpGs showed a sharplypolarized methylation profile being 48.6% hypomethylated(<20% methylation level) and 31.8% hypermethylated(>80% methylation level). We found a significantly higherproportion of piRNA-linked CpGs within the total hypomethylated loci  compared to those found within the hypermethylated loci (p = 1.585E-05). In order toobtain more comprehensive characterization of sperm specific piRNAs-linkedCpG methylation, we performed a comparative analysis with a differentiatedsomatic cell type (B cell) and a colon cancer cell type (HCT116).We were interested in providing a detailed description of the methylationstatus of piRNA-linked CpGs in spermatozoa, B cells and cancer cells. Toaccomplish this purpose, we crossed the position of 15,000 piRNAs withunique positions in the genome present in the piRNABank   . Sperm DWe next focused on the characterization of sperm-specific piRNAs.Accordingly, we identified the sperm-specific hypo and hypermethylated sitesi.e. not overlapping with any of the two other cell types. We performed a GOanalysis for the genes overlapping sperm-specific piRNA in order to definethe type of biological processes in which the associated genes are involved. A totalde novo methylationprocess is followed by a widespread erasure of DNA methylation in primordial germcells. Subsequently, another wave of de novo methylation takesplace during spermatogenesis, ensuring a male germ line specific pattern of DNAmethylation. The understanding of this complex process and the description of spermDNA methylome have multiple implications, including evolutionary The mammalian germ line undergoes extensive epigenetic reprogramming duringdevelopment and gametogenesis. In pre-implantation embryo, a pattern of somatic-likeDNA hypermethylation is established in all cells, including those which are destinedto give origin to germ cells. This active 2 >0.989) and the two subpopulations did not showdistinct clustering of their methylation profiles. However, by analysing theepigenetic distances between the two sperm fractions we were able to detect asignificant difference only in one subject, although the correlation between his twosperm subpopulations was high also in this case, R2 = 0.9896. We separately analyzed a series of genes for whichDNA methylation defects had been previously reported in association with impairedsperm production/quality that included 45 imprinted genes, available on the array,and 10 additional genes selected from the literature. Despite expectation, the DNAmethylation profile of these genes showed no differences in relationship with spermquality. These data indicate that in normozoospermic men, the global DNA methylationprofile is not affected significantly by structural and functional differencesbetween sperm subpopulations. The extensive conservation of the DNA methylationstatus is especially surprising if we consider that differences have been describedalso at the metabolic level of “poor” quality spermatozoa which couldtheoretically influence the DNA methylation process Our first aim was to provide data on sperm DNA methylation profile inquality-fractioned spermatozoa from the same subjects. Human semen is peculiar forthe heterogeneity of its sperm population presenting a number of differentqualitative features that include kinetic, morphological, metabolic andgenetic/chromatin differences. It is for this reason that sperm selection methodshave been developed for assisted reproductive techniques in order to obtain a highlyenriched subpopulation of spermatozoa exhibiting the best structural and functionalcharacteristics, indicative of optimal fertilizing ability. The question whetherthese quality differences between sperm subpopulations are also reflectingmodifications in the DNA methylation pattern has not been addressed so far. In fact,all studies published to date, except for one, focused on either whole semen or justone selected sperm subpopulation. Our analysis of 487,517 CpGs revealed a profoundstability of the sperm DNA methylome without significant differences between spermfractions enriched in “poor” (swim-down) and “good” quality(swim-up) spermatozoa. For all comparisons we obtained surprisingly highcorrelations  show a highly stable sperm methylation profile betweenindividuals. The few moderate smokers (< 10 cigarettes/day) included in the studydid not cluster together, however it remains an important question whether spermmethylome can be altered by heavy smoking or other exogenous factors.The definition of sperm DNA methylation profile between different normozoospermicsubjects derives from a previous observation showing a significant epigeneticvariability in human germ cells. Our aim was to further explore this observationboth by increasing the number of analyzed CpGs  and by comparing different sperm fractions from differentnormozoospermic individuals. Our data, clearly proved that the methylation patternin different individuals showing similar sperm characteristics without contaminatingcells is highly conserved. In fact, the discrepancy with the previous study may wellbe due to a technical issue i.e. to the presence of contaminating somatic cells,which could account for the observed inter-individual differences in the methylationprofile. The highest correlation was found in the selected fraction enriched with“good” quality spermatozoa with RFor the general description and comparative analyses of the sperm DNA methylome, wefocused on the fraction enriched with “good” quality spermatozoa showinga complete lack of significant inter-individual differences. An interesting featureof the “normal” sperm DNA methylome is its highly polarized methylationprofile towards the two extreme of DNA methylation levels: hypomethylation(<20%) and hypermethylation (>80%). We found that 96% ofall CpGs belonged to one of the above categories. Hypo- and hypermethylated lociwere highly conserved in different individuals reaching to a concordance of95% for hypomethylated CpGs and to 83.3% for the hypermethylated ones.These, so called “conserved CpGs” were further analyzed in comparisonwith the relatively few “variable CpGs” (0.3%) present inspermatozoa and with the B cell DNA methylation profile. The qualitative analysis ofhypo-, hyper- and variably methylated regions showed significant differences betweenthe conserved hypomethylated loci and the other two methylation categories; in fact,we observed a significant enrichment with promoters (63.6%) andislands/shores -linked CpGs in the hypomethylated loci. On the other hand, among thehypermethylated and “variable” CpGs there was a significantoverrepresentation of gene body-linked CpGs which, together with intergenic CpGs,build up >60% of all CpGs. The high inter-individual conservation ofhypomethylated loci, especially abundant in promoter regions, suggests that normalspermatogenesis requires strictly controlled methylation levels in specific genepromoters. At this regard, for the first time we provide evidence about anexceptionally high number of “conserved” hypomethylated X and Ychromosome-linked loci which further supports previous predictions on the importanceof sex chromosome linked genes in spermatogenesis and stimulates further research onthe sex chromosome methylation status in pathological conditions An increasing number of studies are converging on the importance of histone-retainedregions in spermatozoa for embryo development. The first study by Hammoud et al.The most relevant finding concerning the comparison between the DNA methylationprofiles of the male germ cell and the B cell, is that only a minority of CpGsshowed differential methylation (4.6%) between the two cell types and wasmainly due to the overrepresentation of hypomethylated loci in spermatozoa. A totalof 3,344 distinct genes were related to sperm-specific hypomethylated CpGs and amongthe strongest associations appeared “DNA methylation involved in gametegeneration” and “piRNA metabolic processes”. Similarly to thegeneral sperm DNA methylome data, those genes (n = 195) whichwere hypomethylated in histone-retained regions were involved in developmentalprocesses  and spermatogenesis. Thedifferent methylation, in respect to the somatic cell, of the promoters ofspermatogenesis genes is in accordance with the well known importance of epigeneticregulation of cell specific functions. The association with developmental genesfurther reinforces the hypothesis about a programmed histone retention inspermatozoa, which would serve for rapid activation of genes involved in embryonicdevelopment.Finally, the complete lack of studies focusing on the methylation status of piRNAs inspermatozoa prompted us to provide a detailed analysis of this specific class ofsmall non coding RNAs. Although piRNAs were first described as specificallyexpressed in the testis, recent data suggest their potential role in tumorigenesisand in somatic cell function In conclusion, our study, based on the largest number of subjects ever considered forsuch a high number of CpGs, provided clear evidence of a highly conserved DNAmethylation profile among normozoospermic subjects. We also demonstrated that spermmethylation is stable in different quality-fractioned sperm subpopulations of thesame individual i.e. sperm methylation is not altered in “poor” qualityspermatozoa of normozoospermic men despite the fact that these cells are clearlydifferent from a metabolic and DNA integrity point of view. In addition, ourarray-based analysis provided both confirmatory and novel data concerning the“normal” sperm DNA methylome, including its peculiar features in respectto somatic and cancer cells. Our description about a highly polarized spermmethylation profile, the clearly distinct genomic and functional organization ofhypo versus hypermethylated loci and the association of histone-enrichedhypomethylated loci with embryonic development, which we now extended also tohypomethylated piRNAs-linked genes, represents a solid basis for future basic andclinically oriented research.Figure S1Comparison of the methylation levels obtained in the array versus datareported in Molaro etal paper (ref 15). Heatmap generated from the distancecorrelation matrix for the 8 individual samples “up” (A) and“down” (B), the scale of the correlation is shown above thematrix ; Correlation scatter plots betweenMolarós data vs the average methylation level for all“up” samples (C) and “down” samples (D), the Pearsoncorrelation coefficient (rho) is shown.(DOC)Click here for additional data file.Table S1Clinical description of the 8 normozoospermic individuals.(DOC)Click here for additional data file.Table S2Analysis of intra-and inter-individual variability of the sperm DNAmethylation profile: estimate of correlation coefficients.(DOC)Click here for additional data file.Table S3Analysis of inter-individual variability of the sperm DNA methylationprofile: epigenetic distance and coefficient of variation.(DOC)Click here for additional data file.Table S4Biological processes associated with genes linked to“conserved” hypo- and hypermethylated CpG loci inspermatozoa.(DOC)Click here for additional data file.Table S5Biological processes associated with genes linked to sperm-specifichypomethylated CpG loci.(DOC)Click here for additional data file.Table S6Biological processes associated with genes linked to piRNAsspecifically hypo or hypermethylated in spermatozoa.(DOC)Click here for additional data file.


Synchronous development of the endometrium (to achieve a receptive state) and of the embryo is essential for successful implantation and ongoing pregnancy. Endometrial receptivity exists only for a finite time in a menstrual cycle and the endometrium is refractory to embryo implantation outside of this window. Administration of hormones to stimulate multifollicular development within the ovary, integral to the majority of assisted reproduction (ART) protocols, dramatically alters the hormonal milieu to which the endometrium is exposed versus normal menstrual cycles. Endometrial maturation may be profoundly affected by this altered endocrine environment.Compare endometrial histology in fertile women, fertile women undergoing hormonal stimulation for oocyte donation and infertile women undergoing fresh embryo transfers in an ART cycle with further comparisons between women who did or did not become pregnant. Examine the presence of leukocytes and markers of endometrial maturation.Endometrial histology was examined by hematoxylin and eosin staining with a semi quantitative scoring method developed to compare histological appearance of tissues. The presence of leukocytes and developmental markers was examined by immunohistochemistry and scored.Endometrial histology was dramatically altered upon stimulation for ART. However, those women who became pregnant presented with significantly less alterations in histological endometrial maturation. Numbers and activation status of leukocyte populations were also altered within the endometria stimulated for ART, with neutrophils undergoing degranulation, usually observed only pre-menstrually.We propose that such developmental changes render the endometrium hostile to the embryo and that modifications to ART protocols should be considered to take account of the requirement for endometrial receptivity and hence increase pregnancy rates. Implantation of the embryo into the endometrium is an essential step to a healthy ongoing pregnancy. A developing embryo can only implant during the short time in each cycle when the endometrium is receptive  cycles to achieve an optimal number of high quality eggs for fertilisation. However, pregnancy rates resulting from fresh embryo transfers still hover around 30% Endometrial receptivity in normal menstrual cycles is still not well defined at a molecular level Histological, immunological, transcriptomic, proteomic and secretomic studies have demonstrated that the ART endometrium presents a different environment to the blastocyst compared with normally cycling endometrium at the same stage of the menstrual cycle a) a comprehensive comparison of the fertile non-stimulated endometrium with stimulated endometria from fertile (donor cycles) and infertile women, b) comparisons between stimulation protocols (GnRH agonist vs GnRH antagonist), and c) comparisons between subjects who subsequently became pregnant and those who did not. A number of parameters were examined: key histological features of the endometrium, the presence of developmental markers (progesterone receptor (PR) and prolactin), the abundance of specific immune cell populations and the localisation and appearance of blood vessels.Previous histological studies of ART endometria have lacked descriptive microscopic images for the cellular changes described and specific markers for developmental changes. Furthermore, the focus has been on a single ART protocol compared with fertile endometria taken at the equivalent stage of the natural cycle, making it difficult to directly compare protocol effects. Therefore, the aims of this study were to present We demonstrate significantly altered endometrial histology in those women who did not become pregnant, with fewer developmental alterations in those who did become pregnant. We also demonstrate altered leukocyte numbers and the presence of activated neutrophils in the endometria of those women who did not achieve pregnancy.Ethical approval was obtained from Institutional Ethics Committees at Southern Health and Monash Surgical Private Hospital for all tissue collections. Written informed consent was obtained from all subjects.This is a retrospective study comparing the endometrial histology of normal fertile women at LH+2 (peak levels measured in urine samples) with women stimulated for ART or oocyte donation at hCG+2 (oocyte pick up). Patients with uterine abnormalities such as leiomyomas, endometrial polyps, or who had received steroid hormone therapy  in the last 6 months were excluded. We studied 57 women undergoing in vitro fertilization (IVF), intracytoplasmic sperm injections (ICSI) or oocyte donation cycles between August 2006 and August 2008. We did not apply a cut off for age, BMI, parity, live births or previous ART cycles . Cumulat2) until the visualization of at least three follicles ≥17 mm when ovulation was triggered with 250 µg recombinant human chorionic gonadotropin (hCG). In this group 13 women subsequently became pregnant and 16 did not become pregnant (retrospective information). Of those who became pregnant (n = 13), 8 underwent in vitro fertilization (IVF) while 5 underwent intracytoplasmic sperm injection (ICSI). Within this group 2 women had endometriosis, 4 women had polycystic ovary (PCO) and 2 women had polycystic ovarian syndrome (PCOS). Of the women who did not become pregnant (n = 16), 1 underwent IVF and 15 underwent ICSI. Within this group, 3 women had PCO.In the GnRH agonist group  oral contraceptive pill (OCP) was commenced between day 1 and day 7 of the preceding cycle for at least 21 days, followed by administration of the GnRH agonist  15 days after commencing OCP. After 10–14 days of GnRH agonist, ovarian stimulation was commenced with 125–225IU recombinant follicle stimulating hormone (FSH) daily  ovarian stimulation was commenced on day 2 of the menstrual cycle with 125–225IU recombinant FSH daily as above until the visualisation of at least one follicle ≥14 mm when administration of GnRH antagonist, cetorelix acetate (0.25 mg), commenced up to and including the day of hCG administration. When at least three follicles ≥17 mm were visualized, ovulation was triggered with 250 µg recombinant hCG. In this group, 6 women underwent IVF while 8 underwent ICSI. Within this group, 2 women had endometriosis. No women in this cohort became pregnant in the cycle of sampling.In the GnRH agonist oocyte donor group (n = 14), fertile women were stimulated via the GnRH agonist protocol (described above) for oocyte donation. These women followed the same protocol as infertile women until the day of hCG administration and oocyte retrieval. These women did not receive luteal phase progesterone.Oocyte retrieval was performed two days after hCG administration (hCG+2) by transvaginal ultrasound guided aspiration. Endometrial biopsies were taken by Pipelle on the day of oocyte collection from GnRH agonist donor cycles, GnRH agonist stimulated infertile women and GnRH antagonist stimulated infertile women. All infertile women from whom a biopsy was obtained underwent a fresh embryo transfer with embryos generated from their own oocytes. Fertile oocyte donors did not undergo an embryo transfer, their hormone treatment ceased at ovulation triggering and their study involvement ceased at endometrial biopsy. The luteal phase of all stimulated embryo transfer cycles was supplemented with vaginal administration of 8% progesterone gel  from day 3 after oocyte collection.et alHuman endometrium was obtained by curettage from normal cycling women 2 days after the LH surge (LH+2: as assessed by urinary assay) following laparoscopic sterilization or assessment of tubal patency . Menstrual cycle stage in normal cycling women was confirmed by histological dating, according to the criteria of Noyes All tissue was fixed in formalin and processed to wax under standardized conditions in the same laboratory.2O). Tissue histology was assessed following hematoxylin and eosin staining.Paraffin sections (5 µm) were dewaxed in Histosol  and rehydrated through descending grades of alcohol (95–70%) to distilled water , 2% normal human serum (‘in-house’) in TBS/0.1% Tween 20  in a humidified chamber for 30 min at room temperature. Primary antibodies were subsequently applied overnight (16–18 h) at 4°C, at the following concentrations; CD56 (1.1 µg/ml), CD68 (2.4 µg/ml), CD45 (1 µg/ml) and NE (1.3 µg/ml). Following washing to remove excess antibody (TBS/0.1% Tween 20), biotinylated horse anti-mouse IgG  diluted in non-immune blocking solution (1∶200) was applied for 30 min. Following stringent washing in TBS-Tween (0.6%), antibody localization was detected using an avidin/biotin peroxidase detection system . Positive localization of leukocytes  was identified by the application of the peroxidase substrate 3, 3′-diaminobenzidine , which produces a brown precipitate. Tissue sections were counterstained with hematoxylin, dehydrated through ascending grades of ethanol (70–95%) and histosol and mounted with DPX. Negative controls were included for each tissue section with substitution of the primary antibody with a matching concentration of mouse IgG  as appropriate.Leukocytes. Leukocyte subpopulations were identified in endometrial tissue using antibodies raised in mouse directed against leukocyte cell markers: total leukocytes , uterine natural killer cells (uNK) , macrophages , and neutrophils , Dako) as described previously PR protein localization was performed following the immunostaining protocol used for CD56, with substitution of the primary antibody by mouse anti-PR (detecting PR-A plus PR-B) applied at 3.6 µg/ml . Biotinylated horse anti-mouse IgG was applied at 1∶200 for one hour prior to detection with ABC-HRP and 3, 3′-diaminobenzidine.CD34 protein localization was performed following the antigen retrieval protocol used for CD56. Sections were blocked with 10% normal rabbit serum, 2% normal human serum (‘in-house’) in TBS/0.1% Tween 20 for 30 min at room temperature. Goat anti-CD34 was applied at 0.5 µg/ml (Serotec) overnight at 4°C. Biotinylated rabbit anti-goat secondary antibody was applied at 1∶200 for one hour prior to detection with ABC-HRP and 3, 3′-diaminobenzidine.No antigen retrieval was performed prior to immunodetection with rabbit anti-prolactin  applied at a 1∶1.75 dilution overnight at 4°C in a 10% normal goat serum, 2% normal human serum, TBS/0.1% Tween 20 blocking solution. Biotinylated Goat anti-rabbit  was applied at 1∶200 for 30 mins prior to detection with ABC-HRP and 3, 3′-diaminobenzidine.Histological and immunohistochemical examination was performed for tissue structure, blood vessel integrity and localization, PR and changes to immune cell populations using an Olympus CH30 microscope. High-resolution images were captured with a Fujix Hc-2000 digital camera. Tissue histology and positive immunostaining was semi-quantitatively scored by two independent experienced observers blind to the nature of the tissue.We applied a histological scoring system based on the similarity of the tissues to the appearance of normally fertile tissues at LH+2  was analyzed and allocated a score from 0–3∶0 (no staining); 1 ; 2 (strong staining); 3 (intense staining). Leukocyte numbers within the endometrium were semi-quantitatively scored, using a scoring system based on the proportion of the stroma occupied by leukocytes (0–100%).All semi-quantitative data is presented as mean ± SEM and was tested for normal distribution. Statistical analysis was performed using ANOVA followed by a Mann Whitney test to determine comparisons between groups. P value <0.05 was taken as significant.The data for patient demographics is presented in Histologically, the non-stimulated fertile endometrium at LH+2 (n = 9) presented with normally organized narrow, straight glands containing no or very few vacuoles . The glaExamination of GnRH agonist-stimulated endometria from fertile oocyte donor cycles revealed extensive sub-nuclear vacuoles within the glandular epithelial cells , arrows.Endometrial tissues from infertile women stimulated with the GnRH antagonist protocol generally presented with highly tortuous, secretory glands , arrows.Infertile women stimulated with the GnRH agonist protocol were subdivided into two groups; those who did or did not become pregnant following the biopsy, allowing correlation of histological changes with pregnancy outcome.Endometrial samples from women who did not become pregnant were heterogeneous in appearance. Some had a secretory phenotype, with sub-nuclear vacuoles present in the glandular epithelium , arrows,In women stimulated with GnRH agonist who subsequently became pregnant, the endometrial histology was less disturbed than in those who did not become pregnant. The glands were generally smaller and more compact . HoweverThus, the histological appearance of the endometrium in the ‘pregnant’ group represents an ‘intermediate’ between the substantially altered histological appearance of the other stimulated groups  and the normal fertile endometrium at LH+2.Immunoreactive PR showed no significant changes upon semi-quantitative analysis, although trends were apparent in staining of luminal epithelium, glands and stroma of stimulated  versus nTotal leukocyte numbers (CD45+) were significantly elevated <0.05 in Total neutrophil number (detected by staining for neutrophil elastase) did not alter significantly with treatment  but bothEndometrial blood vessels were assessed using CD34 staining for endothelial cells . FertileDecidualization of the stroma, demonstrated by prolactin immunostaining, is initiated during the mid-late secretory phase in natural menstrual cycles, specifically close to spiral arterioles. In non-stimulated tissues (LH+2) of fertile women, the stroma was compact with little evidence of edema or prolactin staining . StimulaIn assisted reproduction an embryo that has been fertilized in vitro is replaced into the uterine cavity of the women where it can implant and establish a pregnancy. However, the hormonal regimen used to stimulate ovum development earlier in the menstrual cycle, may disturb endometrial development such that it is not fully receptive for implantation. This paper defines substantial changes in endometrial histoarchitecture and immunocytochemical markers of endometrial differentiation induced by ovarian stimulation protocols in IVF treatment cycles and, importantly, has shown that these disturbances were less severe in women who subsequently become pregnant than in those women who did not. It also demonstrates significant differences in the localization, activation status and number of leukocytes in the endometria of infertile women stimulated for ART, suggesting an enhanced inflammatory environment. This supports and extends previous evidence that the ART endometrium represents an inherently altered tissue when compared with endometria from the natural cycle at an equivalent cycle stage and that this is not conducive to the establishment of pregnancy. While the accuracy and reliability of standard methods for assessing and dating the endometrium based on subtle changes in endometrial histology, has been seriously challenged in a number of studies Previous studies have focused mainly on only one IVF protocol making it difficult to interpret the overall ART protocol effects on endometrial histology. Disparities can arise from the stimulation protocol, the patient selection criteria and the endocrinological parameters Initial comparisons between normally fertile women at LH+2 and fertile women undergoing agonist stimulation for ovum donation demonstrated significant endometrial histological changes. We propose these changes are mediated by the GnRH agonist protocol, which by facilitating multi-follicular development, exposes women to supra-physiological levels of FSH and a higher level of estrogen, in addition to precocious rises in progesterone if ovulation is not appropriately suppressed The infertile GnRH antagonist group at hCG+2 presented a highly heterogeneous collection of samples. While some tissues showed extremely advanced endometria , others The infertile GnRH agonist group at hCG+2 was divided depending on pregnancy outcome. Importantly, while still altered compared with normal fertile women at LH+2, the group who became pregnant had significantly less disturbed endometrial histology than those who did not become pregnant: the latter showed features of secretory changes and evidence of a ‘fragile’ edematous endometrium, such as that often observed in women using hormonal contraceptives In this study we examined progesterone receptors as previous studies have demonstrated significant differences in progesterone receptors between fertile women and those stimulated for IVF via the GnRH antagonist protocol Endometrial leukocytes undergo dramatic changes in both number and relative composition throughout the normal menstrual cycle. Whereas leukocytes are present in relatively low numbers during the proliferative phase of the cycle, these increase through the secretory phase until peri-menstrually they account for around 40% of the cell content of the stromal compartment Pre-decidual cells are normally present in cycling endometrium, only in the late secretory phase It is thus clear that ART endometria at hCG+2, regardless of stimulation protocol or previous fertility status, demonstrate altered endometrial development and that the extent of this disturbance can determine whether or not pregnancy can be established in that cycle. This study does not aim to provide a predictive histological test for endometrial receptivity as the biopsy, tissue processing and immunohistology required are burdensome and unlikely to provide an indication of whether an embryo should be placed into the uterine cavity within the same cycle. Quick assessments of uterine receptivity markers present in a minimally invasive uterine lavage sample It is possible that modifications to current clinical ART stimulation protocols may provide more normal synchronous endometrial development. Recently developed ‘low intensity’-IVF stimulation protocols make controlled ovarian hyperstimulation less complex, cheaper and with reduced chances for adverse complications In conclusion, this paper demonstrates considerable disturbance of endometrial histology in women stimulated for ART, irrespective of fertility status. Importantly, in those infertile women who did become pregnant following stimulation, the endometrium more closely resembled the fertile endometrium at LH+2. We propose that the disturbed endometrial transformation and leukocyte activation status defined here, render the endometrium less or non receptive for embryo implantation, indicating that either modification of protocols or transfer of frozen embryos into natural cycles, may help optimise outcomes for infertile couples.


Most patients with cancers of the nasal cavity or paranasal sinuses are candidates of radiation therapy either due incomplete resection or technical inoperability. Local control in this disease is dose dependent but technically challenging due to close proximity of critical organs and accompanying toxicity. Modern techniques such as IMRT improve toxicity rates while local control remains unchanged. Raster-scanned carbon ion therapy with highly conformal dose distributions may allow higher doses at comparable or reduced side-effects.The IMRT-HIT-SNT trial is a prospective, mono-centric, phase II trial evaluating toxicity (primary endpoint: mucositis ≥ CTCAE°III) and efficacy  in the combined treatment with IMRT and carbon ion boost in 30 patients with histologically proven (≥R1-resected or inoperable) adeno-/or squamous cell carcinoma of the nasal cavity or paransal sinuses. Patients receive 24 GyE carbon ions (8 fractions) and IMRT (50 Gy at 2.0 Gy/fraction).The primary objective of IMRT-HIT-SNT is to evaluate toxicity and feasibility of the proposed treatment in sinonasal malignancies.NCT 01220752Clinical trial identifier  Sinonasal malignancies are a heterogeneous group of tumours of the nasal cavity and paranasal sinuses accounting for about 5% of head and neck tumours and 1% of all cancers . Most coIn view of the rarely complete surgical therapy, most patients will undergo radiation therapy at one point in the course of their treatment. Maximal surgical resection followed by adjuvant conventional radiotherapy leads to a local control of 59% and an overall survival of 40% at 5 years ,7. RecenRecent clinical data though are showing promising results. Hoppe et al treated 37 patients with sinunasal malignancies with postoperative IMRT to a median dose of 60 Gy . This trMucositis ≥ CTCAE°III is a very painful radiogenic acute toxicity leading to significant reduction of the patients' quality of life. In addition, higher grade mucositis may also lead to increased hospital admission for intensive supportive therapy including cost-intensive parenteral feeding. In addition, severe mucositis also leads to an increased number of therapy interruptions or even discontinuation of therapy and hence worsening o therapeutic outcome. In cases where additional chemotherapy is applied concomitantly, it is even more important to keep radiation-induced mucositis to a minimum to maintain compliance with the treatment regimen.Both carbon ion therapy and IMRT have the potential to reduce normal tissue injury while allowing dose escalation within the tumour or prior tumour bed. The present trial (IMRT-HIT-SNT) was therefore designed to evaluate toxicity with special focus on mucositis ≥ CTCAE°III and efficacy in combined intensity-modulated RT and carbon ion boost.The IMRT-HIT-SNT trial is a prospective, non-randomized phase II feasibility trial evaluating acute mucositis ≥ CTCAE°III as the primary endpoint.Based on the fact that local control seems to be dose-dependent and local failure within the high-dose area remains the predominant site of relapse ,21, the To evaluate feasibility and toxicity (with a focus on mucositis CTCAE °III) and efficacy of the treatment.Incidence of mucositis ≥ CTC°III will be assessed as the primary endpoint of the trial, local control, disease-free survival, overall survival, toxicity (incl. mucositis CTCAE °I-II and late toxicity at 2 years post RT).Incidence of mucositis CTC°III in the reported data was 14.1% for IMRT to similar total doses . As desc• Histologically confirmed or incompletely resected adenocarcinoma or squamous cell carcinoma of the nasal cavity or paranasal sinuses• Inoperable tumour or refusal to undergo surgical resection• Macroscopic or microscopic residual tumour (R2/R1) or• ≥T3/T4 or• written informed consent• pts aged 18 - 80 years• effective contraception for pts in childbearing age (<12 months post beginning of menopause)• Prior radio- or chemotherapy for tumours of the head and neck• Other previous malignancy within the past 5 years except prior, adequately treated basal cell carcinoma of the skin or pre-invasive carcinoma of the cervix• Significant neurological or psychiatric condition including dementia or seizures or other serious medical condition prohibiting the patient's participation in the trial by judgement of the investigators• Legal incapacity or limited legal capacity• Positive serum/urine β-HCG/pregnancy• Drug abusePatients are immobilized using individual thermoplastic head masks with thermoplastic shoulder fixation. Planning examinations consist of a planning CT scan (3 mm slice thickness) with the patient positioned in the individual fixation device and contrast-enhanced MRI for 3D image correlation.CTV1 (carbon ion boost) includes the macroscopic tumour, positive lymph nodes and prior tumour bed with special focus on the R1-area. PTV1 consists of a 2 mm margin around the CTV1 but does not extend into critical organs at risk .We prescribe a dose of 24 GyE carbon ions in 3 GyE/fraction (5 fractions per week) to the CTV1, we aim at covering the CTV1 with the 95% prescription isodose. CTV2 includes CTV1 with safety margins along typical pathways of spread as well as the complete surgical operational area. Locoregional nodal areas (levels II and III) are also included within the CTV2, in case of radiologically suspect lympüh nodes, further levels are included as indicated. The CTV2 also takes account for set-up variations, hence corresponds to the PTV2 (CTV2 = PTV2). 50 Gy IMRT (inversely planned step-and-shoot or tomotherapy technique) in 25 fractions (5 fractions per week) are prescribed to the CTV2 (coverage at least with the 90% prescription isodose) taking into account doses applied by daily image guidance with MV-cone-beam CT.Carbon ion therapy treatment planning is carried out using a dedicated treatment planning system (TPS) developed for and in co-operation with HIT(Heidelberg ion therapy centre). TPS offers the following functionalities also expected in conventional radiation therapy as well as methods for biological RT treatment optimization. As ion beams exhibit an increased biological effective dose depending on various factors, these need to be included within the planning algorithm. In addition, steering parameters for scanned ion beams need also be calculated by the TPS.Carbon ion treatment is given at the HIT after inverse treatment planning in active beam application (raster-scanning method). A monoenergetic ion beam with a full-width/half-maximum (FWHM) of 5mm is extracted from the accelerator system (synchrotron) and magnetically deflected to subsequently scan all planned iso-energetic slices roughly corresponding to the tumour's radiological depth. Using this method almost any desired dose distribution can be created.®. IMRT hence is delivered either at a 6 MV-linear accelerator (step-and-shoot technique) or at a 6 MV tomotherapy unit.Intensity-modulated RT is planned at two commercially available planning systems: KonRad (Siemens OS) for step-and-shoot applications or TomotherapyDaily image guidance for carbon ion therapy consists of orthogonal x-ray controls in treatment position, for IMRT image guidance consists of MV cone-beam CTs.Patients do not routinely receive prophylactic feeding tubes, however if they uncommonly experience significant weight loss we will of course offer feeding tube insertion or parenteral feeding.After obtaining written informed consent, patients are included into the trial and receive their treatment planning investigations. Treatment starts with 8 fractions carbon ion (8 × 3 GyE C12) therapy followed by 27/28 fractions of IMRT corresponding to a total dose of approximately 74 GyE  using Enzyme-Linked Immunosorbent Assay (ELISA).• Further, platelet protein content (i.e. tumor angiogenesis growth factors and cytokines) will be analyzed using citrate blood samples and correlated with serum- and plasma- protein results.In order to perform the genomic analysis, patients' blood samples are collected as indicated and RNA, miRNA and DNA isolation will be performed. Based on an established platform, linear RNA-amplification, labelling and hybridization on human genome wide oligo-arrays  are planned. DNA samples are used to identify potential chromosomal aberrations or epigenetic alterations that might predict treatment response. RNA and miRNA samples are further analyzed by real time quantitative RT-PCR to confirm microarray data and to test a subset of clinical predictors.The determinations of proteomic and genomic parameters will be carried out at the Department of Radiation Oncology in Heidelberg. No further genetic investigations on the blood collected during the study will be carried out.Assessment of efficacy will be carried out by evaluation of imaging studies (MRI) at each follow-up. If applicable  tumour response will be evaluated according to the RECIST-criteria . OccurreThe IMRT-HIT-SNT trial has been designed by the Department of Radiation Oncology, University of Heidelberg, and is carried out at the Heidelberg Ion Therapy Centre (HIT). It is an investigator-initiated trial; the Department of Radiation Oncology is responsible for co-ordination, overall trial management, registration , database management, quality assurance, monitoring, and reporting.Patients are recruited by the Department of Radiation Oncology.Adverse and serious adverse events are recorded using NCI common toxicity criteria for adverse events (CTCAE v.4). Acute radiation effects are defined as effects occurring within 90 days from beginning of radiotherapy. Late effects are defined as effects observed thereafter. Safety analysis is performed with respect to frequency of serious adverse events and adverse events stratified by organ system, severity, causality.Patient accrual is completed with inclusion of the last patient and should extend for approximately 4 years from trial initiation. Regular trial participation for each patient terminates 2 years post inclusion into the trial or the patient's death respectively.• Patient wish• Medical condition necessitating treatment termination and withdrawal of the patient from the trial• Pregnancy• Lack of complianceThe trial can be prematurely closed or suspended by the LKP in following cases:• Medical or ethical reasons relevantly affecting the risk-benefit relationship,• Difficulties in recruitment of subjects suggest unjustifiable prolongation of the study timeline,• Previously unexpected adverse events  occur with unjustifiable frequency,• Expected adverse events occur with an unexpectedly high incidence,• Relevant superiority of patients in one treatment arm of a comparable clinical trial,• Legal authorities' decisionThe Ethics Committee (EC) and the competent regulatory authorities will be informed about premature closure of the trial. Furthermore, the Ethics Committee(s) and competent regulatory authorities themselves may decide to stop or suspend the trial.If the trial is closed prematurely, the trial material such as completed, partially completed, and blank CRFs will be returned to the coordinating investigator.All involved investigators have to be informed immediately about a cessation or suspension of the trial. The decision is binding on all trial centers and investigators.The final protocol was reviewed by the ethics committee of the University of Heidelberg Medical School (S-319/2009). The trial complies with the Helsinki Declaration in its recent German version, the Medical Association's professional code of conduct, principles of Good Clinical Practice (GCP) guidelines and the Federal Data Protection Act. It will be carried out in keeping with local legal and regulatory requirements. It is also subject to authorization by the German radiation protection authority (Bundesamt für Strahlenschutz: = BfS). Medical confidentiality and Federal Data Protection Act will be followed. Written informed consent is obtained from each patient in oral and written form.Sinonasal tumours are rare malignancies of the nasal cavity and paranasal sinuses. Preferred treatment would generally be complete surgical resection. Due anatomical site and proximity to critical structures surgical treatment is very complex and clear margins are rarely achieved. Therefore, the majority of patients will undergo RT either because of involved surgical resection margins or technical inoperability.Outcome with adjuvant or definitive RT however has so far been hampered by high rates of accompanying toxicity. Modern radiotherapy techniques such as IMRT and image-guided RT (IGRT) have improved toxicity. Despite these sophisticated new techniques, it remains challenging to apply sufficient doses to the tumour in order to improve control rates.This phase II trial was designed to evaluate the combination of 50 Gy IMRT plus 24 GyE carbon ion boost to a total dose of 74 GyE with respect to toxicity and control rates. To our knowledge, this is the first prospective trial evaluating this treatment regimen in sinonasal cancers.The authors declare that they have no competing interests.MWM, CWK, and JD developed the study protocol and planned the trial. CWK is responsible for statistical considerations/basis of the trial. ADJ, AN, MWM are responsible for conducting and co-ordination of the trial as well as patient recruitment. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/190/prepub


SRY-related homoebox transcription factor 2 (Sox2) as expressed in the embryonic ENS. We now show that Sox2 is expressed in the ENS from embryonic to adult stages and constitutes a novel marker of ENS progenitor cells and their glial cell derivatives. We also show that Sox2 expression overlaps significantly with SOX10, a well-established marker of ENS progenitors and enteric glial cells. We have developed a strategy to select cells expressing Sox2, by using G418 selection on cultured gut cells derived from Sox2geo/+β mouse embryos, thus allowing substantial enrichment and expansion of neomycin-resistant Sox2-expressing cells. Sox2geoβ cell cultures are enriched for ENS progenitors. Following transplantation into embryonic mouse gut, Sox2geoβ cells migrate, differentiate, and colocalize with the endogenous ENS plexus. Our studies will facilitate development of cell replacement strategies in animal models, critical to develop human cell replacement therapies for HSCR. Stem Cells 2011;29:128–140The capacity to identify and isolate lineage-specific progenitor cells from developing and mature tissues would enable the development of cell replacement therapies for disease treatment. The enteric nervous system (ENS) regulates important gut functions, including controlling peristaltic muscular contractions, and consists of interconnected ganglia containing neurons and glial cells. Hirschsprung's disease (HSCR), one of the most common and best understood diseases affecting the ENS, is characterized by absence of enteric ganglia from the distal gut due to defects in gut colonization by neural crest progenitor cells and is an excellent candidate for future cell replacement therapies. Our previous microarray experiments identified the neural progenitor and stem cell marker  During embryonic development, multipotential progenitor cells give rise to the diverse array of tissue-specific cell types found in adult structures. As development proceeds, however, there is a progressive restriction of cells to particular lineages and within a particular lineage to the differentiated cell types that constitute the mature tissue or organ. Nevertheless, some cells retain multipotential progenitor potential late into embryogenesis, at postnatal stages, and even into adulthood . Cells wThe enteric nervous system (ENS) is the part of the peripheral nervous system (PNS) that controls key aspects of gut function, including peristalsis, the regulation of blood flow, and secretion of water and electrolytes. The mature ENS is composed of neurons and glial cells organized as ganglia within in two concentric rings, the myenteric and submucosal plexus, situated between smooth muscle layers and comprises 1%–5% of cells in the gut –4. One oWnt1Cre;Rosa26eYFP NCC lineage marker  box family transcription factors)  mice exhibit aganglionosis of the distal colon, due to loss of ENS progenitors [Sox2 heterozygous mice (such as Sox2geo/+β) are viable and have an apparently normal ENS (data not shown and supporting information Sox2−/− embryos die at implantation stages [Sox2 hypomorphic mutations [Sox2 mutations [Sox10[+/−] mice do not have obvious defects in gliogenesis, Sox10[+/−];Sox8[+/−] mice exhibit apparent reductions in glial cell number [Sox2 would be of great interest.gal NCCs , 48–50, genitors , 51. Desn stages . Furtherutations  or condiutations . Finallyl number . WhetherCritical first steps in developing human cell replacement therapies for HSCR are to establish methods to identify and characterize ENS progenitors, and to use animal models to study the potential of these cells on transplantation. A number of techniques have been developed to isolate ENS progenitors from murine or human sources, including selection based on cell surface marker expression, cell culture techniques that favor growth of progenitors, and selection on the basis of proliferative capacity , 17. A lSox2-expressing population to be selected. Such cells can be expanded and passaged in culture for months, and thus provide ENS progenitors in large numbers. Selection on the basis of Sox2 expression has the added benefit of limiting the number of neurons present in the population, thus reducing the heterogeneity of the population. The usefulness of this strategy in obtaining cells with appropriate progenitor properties is demonstrated by the fact that cells transplanted into the gut can migrate, differentiate, and establish close associations with the endogenous ENS. Moreover, such cells are capable of colonizing aganglionic gut regions. Recent experiments using a similar approach to select embryonic cells on the basis of Sox10 expression has also successfully enriched for cells with migratory and differentiation potential [The approach we describe here attempts to circumvent previous limitations. Having identified SOX2 as a marker of enteric progenitor cells, we combined genetic and cell culture techniques to enable a otential . We suggSox2geoβ cells transplanted into embryonic gut can migrate extensively, differentiate appropriately, and appear to be integrated into the endogenous ENS ganglia. Thus, Sox2geoβ cells also exhibit key properties of ENS progenitors, further supporting their potential use in modeling cell replacement therapies in animal models, with eventual implications on treatment of HSCR.We have identified SOX2 as a novel marker of enteric progenitor cells and glial cells, a finding that will facilitate the study of ENS progenitors. In addition, we have used a selection technique to enrich for SOX2-expressing ENS progenitors among cultured gut cells. This method enables ENS progenitors to be obtained in the large numbers required for studying their properties following transplantation, and to develop cell replacement therapies using animal models. Our experiments show that


Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity. Mice were first sensitized with ovalbumin, extracts of cockroach or house dust mite followed by a challenge period of seven weeks. Further to this, an additional group of mice was sensitized with all three allergens and then challenged with allergen alternating weekly between allergens. All three allergens applied separately to the mice induced comparably strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling, which was characterised by fibrosis and increased smooth muscle thickness. In contrast, application of all three allergens together resulted in a greater Th2 response and increased airway hyperreactivity and a stronger albeit not significant remodeling phenotype compared to using HDM or CRA. In this triple allergen model dexamethasone application, during the last 4 weeks of challenge, showed no suppressive effects on any of these parameters in this model. In contrast, both TLR7 agonist resiquimod and TLR9 agonist CpG-ODN reduced allergen-specific IgE, eosinophils, and collagen I in the lungs. The TLR9 agonist also reduced IL-4 and IL-5 whilst increasing IFN-γ and strongly IL-10 levels in the lungs, effects not seen with the TLR7 agonist. However, neither TLR agonist had any effect on airway hyperreactivity and airway smooth muscle mass. In conclusion we have developed a severe asthma model, which is steroid resistant and only partially sensitive to TLR7 and TLR9 agonist treatment. This model may be particular useful to test new potential therapeutics aiming at treating steroid resistant asthma in humans and investigating the underlying mechanisms responsible for steroid insensitivity. Severe asthma is characterised by persistent inflammation of the airways, airway hyperreactivity and pronounced airway remodeling, involving increased fibrosis and smooth muscle thickening, mucus overproduction and sub epithelial thickening. Patients with this asthma phenotype often exhibit a steroid resistance and are prone to acute exacerbations, leading to the highest mortality rates. These patients account for approximately 30% of the overall healthcare costs caused by asthma Murine models display several hallmarks of severe atopic asthma seen in patients. These include Th2 driven pulmonary inflammation with strong lung eosinophilia, elevated levels of Th2 cytokines such as IL-4, IL-5 and IL-13, the production of allergen specific IgE, airway hyperreactivity, and airway remodeling characterized by goblet cell hyperplasia, mucus over production, smooth muscle thickening and sub epithelial fibrosis The aim of our study was to identify allergens and an immunization protocol which induces severe asthma in mice, ideally showing a phenotype which is insensitive to steroid treatment. For this purpose BALB/c mice were first sensitized with ovalbumin (OVA), extracts of cockroach (CRA) or house dust mite (HDM) together with alum followed by a challenge period of seven weeks in which mice received the respective allergen via intratracheal administration twice a week. Strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling was observed for all three allergens with no major differences between the allergens. In contrast, when mice were immunized with all three allergens simultaneously and then challenged with allergen  a greater Th2 response and increased airway hyperreactivity compared to only single allergen was observed. Fibrosis and smooth muscle thickening was enhanced but not significantly compared to HDM or CRA. Furthermore we were interested if this strong phenotype could be supressed by therapeutic intervention. We chose to use dexamethasone, TLR7  and TLR9 (CpG-ODN) agonists because all three have been previously shown to have potent anti-allergic effects in animal models of asthma Female BALB/cAnNCrl mice  were maintained under conventional conditions in an isolation facility. At the onset of the experiments, all animals were between 6 and 8 weeks of age.All experiments were performed according to the guidelines of the local and government authorities for the care and use of experimental animals .Dermatophagoides pteronyssinus, Greer Laboratories, Lenoir, N.C., USA), and CRA  were solubilised in 100 µl phosphate buffered saline and absorbed to 100 µl Al(OH)3 . For challenge, allergens were solubilised in 50 µl phosphate buffered saline  and administered via intratracheal application.For intraperitoneal sensitization OVA , HDM , 1 hour prior to allergen challenge twice weekly  performed by flushing lung and airways twice with 0.8 ml PBS containing 0.6 mM EDTA and 5 mg/ml protease inhibitor .BAL cells were differentiated after centrifuging 200 μl of BAL fluid on cytospin slides for 1 min at 500 rpm. For differentiation of cells, slides were stained with May-Gruenwald/Giemsa. Cell types were differentiated and counted using light microscopy applying standard morphological criteria. Total counts of cells in BAL fluid were determined using an automated haematology analyzer .Serum levels of allergen specific IgE were measured with ELISA technique on MSD platform using rat anti-mouse IgE  and MSD sulfo-tag-labeled goat anti-rat-antibody . In brief, plates were coated with the respective allergen extract (20 µg/ml) and incubated at 4°C over night. Serial dilutions of serum samples were applied and incubated at 4°C over night. Rat anti-mouse IgE (diluted 1∶200) was added and incubated for 45 min at room temperature. Plates were then washed three times before MSD goat anti-rat antibody (diluted 1:500) was added and incubated for 1 h at room temperature. For allergen specific IgE-level determination, plates were analyzed with MSD Sector Imager 6000 . Levels of allergen specific IgE are given in arbitrary units/ml (U/ml), directly correlating to absorbance.Lung samples were homogenized with FastPrep-24  using PBS containing 5 mg/ml protease inhibitor .Chemokines and cytokines were measured in supernatants of homogenized lung tissue using mouse TH1/TH2 9-PlexBase cytokine/chemokine multiplex assay , IL-13 was measured using mouse ELISA Kit , and total TGF-β 1 was measured with mouse TGF-ß1 Platinum ELISA , all according to manufactureŕs instructions.2)/void area ( µm2) x 100. The void area is the area of the main bronchus free airway lumen.After lavage, the left lung of each mouse was fixed in 4% formalin for 24 h and embedded in paraffin wax. All histology samples were prepared from the same position, where the main bronchus enters the lung. Lung sections (2–3 µm) were stained with haematoxylin and eosin (H&E) reagent  for detection of inflammatory infiltrates, with periodic acid-schiff  for goblet cells and mucus production. For visualising smooth muscle, α-smooth muscle actin antibody  was used. For visualizing collagen, a picro-sirius red staining with picric acid  was performed. Cross sections showing the main bronchus were used for quantification of pathological changes. For quantification of smooth muscle thickening and mucus production automatic analyses were performed using Halcon software . Airway smooth muscle thickness was quantified by assessing diameter of the muscle layer surrounding the main bronchus. In brief, an inner line at the border of airway space (lumen) and epithelium was drawn. In 300 µm distances to the inner circle a second circle surrounding the main bronchus area was drawn. Following this, vertical auxiliary lines in intervals of 2 µm were drawn from the inner to the second circle. For quantification, the length of stained auxiliary line was measured. Following the mean ± standard error of means (SEM) of all measured lengths were calculated. For quantification of mucus, epithelial layers of the PAS stained lung slices were defined by delimiting the area from lumen to sub epithelial tissue. Following this step, areas of epithelial layers and total red stained areas were quantified. The ratio of mucus and bronchus area was calculated. Mucus plugging was scored manually by semi-quantitative analysis (double-blinded). The left lung of a mouse was considered plugged when more than 75% of the main bronchus was filled with mucus. Levels of fibrosis are expressed as collagen to void area in %, a value calculated as follows: sub epithelial collagen area ( µm2O].Airway hyperreactivity (AHR) to methacholine was assessed using whole-body-plethysmography  as described previously Collagen levels were assessed in supernatant of homogenized lung tissue using ELISA technology based mouse type I and III collagen detection kits  according to manufacturer`s instructions.Data analyses were performed using the Prism 4 software package . If not stated otherwise, One-way analysis of variance (ANOVA) with the Dunnett’s-post test was used to determine statistical difference to reference group (negative control or untreated). A P value 0.05 was considered statistically significant.In order to develop a preclinical model of severe human asthma BALB/c mice were treated either with OVA, extracts of CRA, HDM , or a coCompared to using the single allergens mice treated with the combined allergens showed highest baseline pulmonary resistance and lowest baseline compliance. After methacholine challenge these mice also showed the highest increase in resistance . ResistaThe wet weight of the left lung showed significant increase in all treated mice with the highest increase detected in the group treated with all three allergens. The highest cellular influx into lung tissue was also observed in the combination model . In addiDexamethasone and TLR7 and –9 agonists were applied twice weekly, one hour before allergen challenge. Therapeutic application of dexamethasone had no suppressive effects on any of the parameters measured in the combination model  and 6. IThe aim of our study was to identify an immunization protocol which causes aspects of a severe asthma phenotype in mice in order to be able to test new therapeutics within a reasonable time frame and with a sufficiently robust assay window. We directly compared the use of OVA, HDM and CRA which, for sensitization, were applied intraperitoneally together with alum. One of the reasons we chose this strategy versus a lung allergen only challenge model was that we have compared the remodeling phenotype in mice between OVA/alum + OVA lung challenge versus OVA lung challenge only. We found that the intraperitoneal OVA/alum sensitization leads to a much stronger remodeling phenotype in a shorter period of time in comparison to a mucosal sensitization (data not shown). A recent publication supports our finding that intraperitoneal allergen sensitization together with alum induces a much stronger allergic inflammation in the lung compared to lung challenge only When mice were immunized with all three allergens simultaneously and then challenged with allergen  a stronger remodeling, albeit not significant phenotype, greater Th2 response and increased airway hyperreactivity compared to only using the single allergens was observed. Our experiments were not designed to address the question of whether triple allergen combinations are generally better at inducing Th2 responses than when using a single allergen. This clearly depends on the model used, the degree of endotoxin contamination in the allergen preparations, the amounts of allergen applied and other factors. However, using our protocol, triple allergen combination is clearly superior to the use of single allergens alone when analysing all parameters. A recent report has also shown that combining allergens in one asthma model can induce severe disease and this was associated with stopping the development of allergen-induced tolerance A surprising finding was that although the triple combination induced the strongest Th2 response in the lung with the highest IL-13 levels detected (known to directly induce mucus production and goblet cell metaplasia) we did not observe the highest goblet cell hyperplasia and amounts of intracellular mucus in the main bronchus. However, mice in the combination model showed the most severe mucus plugging. The lower amount of mucus staining in the combination group may be explained by an increase in mucus secretion into the airways leading to the observed increased plugging.Mice treated with all three allergens also showed the highest airway hyperreactivity and airway constriction at baseline. This is most likely due to the mucus plugging and enhanced levels of IL-13, increased smooth muscle mass and increase in fibrosis, as all these factors have been shown to contribute to AHR Overexpression of TGF-β1 in mice was shown to cause lung fibrosis Why does the combination of three allergens lead to a stronger Th2 response than when using only one allergen? One reason may be that an increased amount of epitopes for Th2 cell activation is present inducing the stronger Th2 responses observed. The strong increase in IL-2 in the BAL supports the view of stronger T cell activation. IL-2 may also be directly responsible for the enhanced Th2 response as shown previously A major problem for patients suffering from severe asthma is the reduced efficacy of first line steroid treatment, leading to higher dosing or insensitivity to treatment. However, at present there are no allergic asthma mouse models reported reflecting these clinical findings. Published studies using OVA Although, the amount of dexamethasone applied reduced the OVA-induced Th2 inflammation in a chronic setting but not when using the triple allergen model, we cannot conclude that is due to using all three allergens. When we used HDM or CRA a stronger inflammation and remodeling phenotype was also induced compared to when using OVA. It is possible that models using CRA and or HDM may also be dexamethasone resistant.Nippostrongylus brasiliensis was mediated by IL-10 In contrast to the lack of suppressive effects of dexamethasone we found that both the TLR7- and TLR9 agonist were able to reduce some features of the asthmatic phenotype. The TLR7 agonist reduced the influx of eosinophils and allergen-specific IgE levels in the serum. The TLR9 agonist had stronger effects on these parameters and in addition reduced mucus production in the lung and IL-4 and IL-5 levels in the BAL. Neither of the TLR agonists was able to reduce the AHR or the increase in smooth muscle mass. The TLR9 agonist treated mice showed an increase in lung neutrophilia, IFN-γ, IL-1ß and IL-12 levels in the homogenate, indicative of increased Th1/innate responses, suggesting that the TLR9 mediated suppressive effects on the Th2 response may be due to enhanced Th1 or innate responses as discussed previously Taken together we show that using a triple allergen combination with OVA, CRA and HDM for sensitization and allergen challenge, leads to a very severe human asthma like phenotype in the lungs of mice stronger than when using the allergens alone. The phenotype induced reflects the atopic phenotype with a strong Th2 plus weaker Th1 response. This chronic model is steroid insensitive and less sensitive to therapeutic intervention with the TLR7 and TLR9 agonist resiquimod or CpG-ODN than other published severe asthma models Figure S1Histological sections of the lung of mice treated with dexamethasone, TLR7 or TLR9 agonists in triple allergen treated mice (TAC). Representative sections of haematoxilin/eosin (H&E) (A), periodic acid-Schiff (PAS) (B), and sirius red staining of the main bronchus with surrounding area (C) are shown. Scale bar  =  100 µm. Shown are representative sections of 8-12 mice per group. Triple allergen combination (TAC) group.(TIF)Click here for additional data file.Figure S2Dexamethasone reduces OVA induced cellular influx in a chronic model. Mice were treated with OVA as described in P<0.05; **P<0.01; ***P<0.001 in comparison to the PBS control mice and §P<0.05; §§P<0.01, §§§P<0.001 in comparison to the OVA group.(TIF)Click here for additional data file.


Bone disease in patients with juvenile idiopathic arthritis (JIA) is associated with focal (joint erosion and juxtaarticular osteopenia) and systemic bone loss . Pathophysiology of bone loss is multifactorial and involves particularly proinflammatory cytokines and deleterious effects of glucocorticoid therapy. Clinical studies in patients with JIA indicate excessive activation of osteoclastogenesis and reduction of bone formation. Reduction of physical activity, muscle atrophy caused by high disease activity, and compulsory restriction in movements are also associated with bone loss. In patients with JIA, the disease can be complicated by growth cartilage involvement and systemic or local growth retardation. In the absence of preventive measures, fragility fractures can occur even at an early age. Juvenile idiopathic arthritis (JIA) is a systemic autoimmune chronic inflammatory joint disease beginning until 16 years of age. JIA is the most frequent rheumatic systemic disease in the childhood. In the industrial countries, the incidence of JIA is 5–18 and prevalence of JIA is 30–150/100 000 children until 16 years old. In the Czech Republic, the annual incidence of JIA is 13/100 000 and prevalence of JIA is 140/100 000 children until 16 years old [During childhood and adolescence in patients with JIA, when the peak of bone mass is attained in the healthy people, the accrual of bone mass is suppressed through direct and indirect mechanisms, namely by the inflammatory disease, by drug therapy and immobilization , 3. In JTherapy with glucocorticoids can prevent the acquisition of an optimal peak bone mass in young patients. The lower peak of bone mass is associated with the increased risk of osteoporosis and the increased risk of fracture in the adult age –15. Drug therapy with glucocorticoids may increase the risk of developing osteopenia and osteoporosis. Vertebral collapse is more common in children receiving a cumulative dose of at least 5 g of prednisone equivalents, prolonged periods of bed rest, and with low BMD and low serum concentrations of 25-hydroxyvitamin D , 16. In The increased risk of fracture has been proven especially in patients with erosive arthritis, growth retardation and high cumulative dose of glucocorticoids , 3. α) stimulate protein degradation, inhibit myocyte differentiation, and cause myocyte apoptosis [Myopathy caused by the autoimmune inflammation is one of reasons of the low lean mass in patients with JIA. Proinflammatory cytokines  markedly depends on the muscle force affecting the skeleton , 18. RedThe important difference between JIA and rheumatoid arthritis in adults lies in growth retardation of children with JIA, deceleration of growth rate, low stature in adult age and local growth retardation at sites of joints involved in arthritis , 3. A siβ) accelerates degradation of insulin-like growth factor I binding protein 3 (IGFBP-3) resulting in reduction of IGF-I levels and growth retardation. Chondrocyte apoptosis induced by the tumor necrosis factor α (TNFα) through the medium Fas-associated death domain (FADD) acts the important part in growth abnormalities [The growth hormone (GH) and insulin-like growth factor I (IGF-I) are the most important regulators of postnatal growth. In children with JIA and serious growth retardation, the normal pulsative secretion of growth hormone, but low levels of IGF-I were described (resistance to growth hormone). Proinflammatory cytokines influence linear growth of children by their systemic effects and by local effects on growth cartilage of long bones . Increasmalities . Growth malities .Studies of bone formation and bone resorption markers are not unambiguous; however, most studies indicated prevalence of bone resorption over bone formation , the othα, IL-1) produced by synovial membrane are responsible for excessive bone resorption also in adult patients with JIA [In prepubertal children with active JIA, prevalence of bone resorption markers over reduced markers of bone formation corelated with laboratory indices of disease activity, namely in children with polyarticular phase of JIA , 28, 29.with JIA .α (TNFα), interleukin 1 (IL-1), interleukin 6 (IL-6) a interleukin 17 (IL-17), present in arthritic joint can cause an excessive osteoclastogenesis [α significantly elevates bone resorption [α antibodies demonstrably reduces joint erosions [Proinflammatory cytokines, such as tumor necrosis factor ogenesis –35. TNFαsorption  and attesorption –40. IL-1sorption . In patierosions . IL-17, erosions , 42, 43 erosions , 44–46.ŔANKL) have essential importance for osteoclastogenesis and osteoclast function [Receptor activator nuclear factor kappa B (RANK) and its ligand , stimulate production of inhibitors of the Wnt proteins signalling pathway, especially sclerostin and Dickkopf 1 (DKK-1) and consequently inhibit osteoblast differentiation [The extent of the bone involvement in the rheumatic inflammatory processes depended on age and bone maturity . In chilntiation –63  family transcription factors to promote specific gene expression [The Wnt proteins are large family of factors that bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of pression . Decreaspression . The DKKpression , 64. Thepression .Matrix metalloproteinases (MMPs) are responsible for cartilage destruction and periarticular bone erosions in juvenile idiopathic arthritis , 66–68. In children, a negative correlation between bone mass and cumulative dose of glucocorticoids was found –71. DuriMethotrexate, which is the most frequent DMARDs in children, can cause osteopenia in children patients with malignancies, but low-dose methotrexate used in inflammatory diseases did not influence negatively the bone mass , 73.α antibodies was also documented upon the skeleton. Simonini was the first to demostrate increased bone mass after 1-year etanercept treatment in children with JIA; reduction of bone loss was associated with therapeutic response with decreased disease activity [Biological treatment with infliximab and etanercept in children with JIA is associated with decrease in disease activity –76. The activity . EtanercAminobisphosphonates represent an effective option in patients with documented prevalence of bone resorption over bone formation . In JIA,Growth hormone was effective in stimulating collagen production and improving liner growth in JIA –83. HoweIn adult patients with closed linear growth and severe osteoporosis, an intermittent administration of PTH 1–34 (teriparatide) or PTH 1–84 represents an effective option to restore bone structure that has previously been lost .Well-timed and efficient treatment of JIA in children and adolescents can improve bone status. However, patients who suffered from JIA during childhood and adolescence may attain decreased bone mass and have an increased risk of fragility fractures. It is important to identify the subjects with an increased risk of fracture as early as possible. In adult patients with closed linear growth, it is necessary not only to reduce bone resorption, but also to support formation of new healthy bone mass. Several new therapeutical procedures are under investigation.


Chikungunya (CHIKV) has recently seen a re-emergence in India with high morbidity. However, the epidemiology and disease burden remain largely undetermined. A prospective multi-centric study was conducted to evaluate clinical, epidemiological and virological features of chikugunya infection in patients with acute febrile illness from various geographical regions of India.A total of 540 patients with fever of up to 7days duration were enrolled at Karnataka Institute of Medical Sciences (KIMS), Karnataka (South); Sawai Man Singh Medical College (SMS) Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (North) from June 2008 to May 2009. Serum specimens were screened for chikungunya infection concurrently through RT-PCR and serology (IgM). Phylogenetic analysis was performed using Bioedit and Mega2 programs. Chikungunya infection was detected in 25.37% patients by RT-PCR and/or IgM-ELISA. Highest cases were detected in south (49.36%) followed by west (16.28%) and north (0.56%) India. A difference in proportion of positives by RT-PCR/ELISA with regard to duration of fever was observed (p<0.05). Rashes, joint pain/swelling, abdominal pain and vomiting was frequently observed among chikungunya confirmed cases (p<0.05). Adults were affected more than children. Anti-CHIK antibodies (IgM) were detected for more than 60days of fever onset. Phylogenetic analysis based on E1 gene from KIMS patients (n = 15) revealed ∼99% homology clustering with Central/East African genotype. An amino acid change from lysine to glutamine at position 132 of E1 gene was frequently observed among strains infecting children.The study documented re-emergence of chikungunya in high frequencies and severe morbidity in south and west India but rare in north. The study emphasizes the need for continuous surveillance for disease burden using multiple diagnostic tests and also warrants the need for an appropriate molecular diagnostic for early detection of chikungunya virus. Alphavirus of the family TogaviridaeChikungunya virus (CHIKV) is an enveloped positive-strand RNA virus belonging to genus Aedes tayloriAedes aegyptiAedes aegypti and Aedes albopictus are known to exist and are widely prevalent during the post monsoon season, CHIKV was first detected in 1963 in West Bengal Historical data report the first detection of chikungunya in 1952 in the Makonde Plateau in Africa Aedes albopictusCHIKV being an RNA virus is susceptible to high mutation rates which may help the virus to evade the immune response and thus adapt efficiently. However, phylogenetic analysis of E1 gene of CHIKV indicates only three lineages with distinct genotypic and antigenic characteristics i.e. the “Central/East African genotype”, the “Asian genotype” and the “West African genotype” The high morbidity and loss in daily activity associated with CHIKV infection results in considerable economic loss among the affected nations, specifically India st June, 2008 through 31st May, 2009. Patient enrolment was carried out year round at Karnataka Institute of Medical Sciences (KIMS), Hubli, Karnataka (south); Sawai Man Singh Medical College (SMS) Jaipur, Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (north) with AIIMS as the coordinating and testing centre.Patients were recruited at three distantly located regions of India  from 1Patient enrolment involved those with fever ≤7 days duration at the time of their visit to the Out/Inpatient departments at AIIMS (n = 178), KIMS (n = 233) and SMS (n = 129) hospitals and those who were willing to participate in the study (n = 540). The study was approved by each Institution's Ethics Committee . All patients (or for children parents or guardians) gave written informed consents. Demographic details, clinical data including symptoms and onset and duration of fever were recorded in the specified proforma. Approximately 3 ml of blood was collected from each patient enrolled at the time of admission. Convalescent phase (15–90 days of fever onset) sample could be collected from 30 patients only at KIMS center. Sera were separated from the blood by centrifugation and stored at −70°C in aliquots till further use Genomic viral RNA from CHIKV infected culture-lysate/patient sera were extracted using QIA Amp Viral RNA minikit  according to manufacturer's protocol. The 294b E1 gene fragment of CHIKV was amplified with Qiagen one Step RT-PCR kit as described previously All serum specimens were screened for CHIKV specific IgM antibodies by ELISA using a commercial kit  according to manufacturer's recommended procedure.5′-TACCCATTCATGTGGGGC-3′) and reverse (5′-TGACTATGTGGTCCTTCGGGGG-3′) primers. Amplification product was gel purified by the Gene-Clean purification method  and sequenced using a previously described method Sequences were determined from the 913 bp E1-amplicon (nt 10246–11158) that had been generated following RT-PCR (n = 15) with forward .CHIKV infection was tested both for the presence of anti-CHIKV IgM antibodies by ELISA and CHIKV RNA by RT-PCR. A total of 137 (25.4%) subjects tested positive for CHIKV through either of the two tests. A solitary case was detected at AIIMS while relatively higher percentages were observed at SMS (16.3%) and KIMS (49.4%) . Rate ofChikungunya detection was performed by both RT-PCR and IgM-ELISA with detection rates ranging from 4.3%–90% for RT-PCR and 10%–95.6% for ELISA depending on the days of fever onset . This diAdditionally 30 cases from KIMS were assessed for persistence of IgM during convalescence (15–90 days of fever onset). While majority of the chikungunya positive cases (9 of 11) were detected anti-CHIK IgM between 15–90 days post illness, none of the chikungunya negative patients (n = 19) were detected the same (data not shown).Comparison of clinical features between chikungunya positive and negative patients demonstrated rashes, headache, joint pains, joint swelling, abdominal pain, cough and vomiting to be significantly associated with CHIKV confirmed cases (p<0.05) whereas differences for other symptoms were not found significant .We compared the rate of CHIKV prevalence based on patient age groups 0–5, >5–18 and >18 years of age and observed significantly higher detection (p<0.05) among adult populations (aged >18 years). An apparent increase in frequency of CHIKV detection with age, from about 16% cases among children to 50% among adults was observed .In addition Chi square analysis revealed significant differences for presence of rashes, headache, joint pain and joint swelling (p<0.05), between children (<18 years) and adults (>18 years) . InteresComparative sequence analysis of chikungunya E1 gene from 15 CHIKV infected patients from KIMS revealed 98.3%–99.9% homology among them, clustering within the Central/East African genotype . FurtherThe present multi-centre study confirms and extends the findings of recent reports from India and other parts of world indicating a re-emergence of severe chikungunya disease which is becoming a major public threat in India Amongst the three centres, AIIMS and SMS had a relatively higher male∶female ratio. However this could be attributed to the social bias generally observed against females in the north and west India rather than to disease susceptibility based on gender. Various other studies conducted worldwide have shown inconsistencies on gender bias to disease susceptibility with most studies reporting each gender to be equally susceptible Discrepancies in the symptoms of Chikungunya among suspected cases across different studies warrant the requirement of a clear and well defined clinical profile of the disease. For example our study detected rashes more frequently among CHIKV cases as compared to other Our study also demonstrated a link between chikungunya detection rate and geographical location of study centre. We observed highest percentage of cases at KIMS which is located along the coast in the southern part of the country compared to the other two centres that are landlocked regions with extended periods of dry summer and extreme winter. This is in agreement with findings of other studies those reported the prevalence of chikungunya mostly in the coastal regions including South India and South East Asia One interesting observation made in the present study was the near absence of chikungunya case at AIIMS site despite reports of dengue prevalence Aedes albopictus and Aedes aegypti. In India the first CHIKV outbreak in 1963 was observed during July to December, coinciding with the monsoon and post monsoon seasons. However, in the present study, CHIKV was detected throughout the year at KIMS centre although with higher rate during the monsoon season, a situation similar to dengue Chikungunya fever has been observed to occur mostly during the monsoon and post monsoon seasons during which time there is high breeding of both Our study has confirmed the findings of various other studies that certain symptoms like rashes, headache, joint pain/swelling and abdominal pain are significantly associated with chikungunya infections Our finding on higher detection rate of chikungunya among adults as compared to children confirms most of those reported worldwide In addition to assessing clinical and epidemiological implications of chikungunya we tried to identify the molecular composition of the infecting strains. High nucleotide and amino acid homology was observed among the infecting strains which clustered within the Central/East African genotype confirming earlier reports that all recent CHIKV outbreaks in India have been a result of the Central/East African genotype In conclusion, to our knowledge this is the first multi-centre study undertaken to determine the epidemiology of chikungunya fever across a wide age group with year round enrolment of large number of patients simultaneously in three different geographical regions of India. Both genomic and serology based assays were employed throughout the study for confirmation of CHIKV. Our study clearly demonstrates high incidence of CHIKV in India particularly in the southern region with moderate prevalence in the western and rare in the northern region. This warrants continuous CHIKV surveillance in India to determine the disease burden for improved healthcare.