OBJECTIVE:To test the hypothesis that specific proteins and peptides are expressed differentially in eutopic endometrium of women with and without endometriosis and at specific stages of the disease (minimal, mild, moderate, or severe) during the secretory phase.DESIGN:Patients with endometriosis were compared with controls.SETTING:University hospital.PATIENT(S):A total of 29 patients during the secretory phase were selected for this study on the basis of cycle phase and presence or absence of endometriosis.INTERVENTION(S):Endometriosis was confirmed laparoscopically and histologically in 19 patients with endometriosis of revised American Society for Reproductive Medicine stages (9 minimal-mild and 10 moderate-severe), and the presence of a normal pelvis was documented by laparoscopy in 10 controls.MAIN OUTCOME MEASURE(S):Protein expression of endometrium was evaluated with use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. The differential expression of protein mass peaks was analyzed with use of support vector machine algorithms and logistic regression models.RESULT(S):Data preprocessing resulted in differential expression of 73, 30, and 131 mass peaks between controls and patients with endometriosis (all stages), with minimal-mild endometriosis, and with moderate-severe endometriosis, respectively. Endometriosis was diagnosed with high sensitivity (89.5%) and specificity (90%) with use of five down-regulated mass peaks (1.949 kDa, 5.183 kDa, 8.650 kDa, 8.659 kDa, and 13.910 kDa) obtained after support vector machine ranking and logistic regression classification. With use of a similar analysis, minimal-mild endometriosis was diagnosed with four mass peaks (two up-regulated: 35.956 kDa and 90.675 kDa and two down-regulated: 1.924 kDa and 2.504 kDa) with maximal sensitivity (100%) and specificity (100%). The 90.675-kDa and 35.956-kDa mass peaks were identified as T-plastin and annexin V, respectively.CONCLUSION(S):Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis of secretory phase endometrium combined with bioinformatics puts forward a prospective panel of potential biomarkers with sensitivity of 100% and specificity of 100% for the diagnosis of minimal to mild endometriosis.

Orthopedic implants are categorized into two groups including permanent joint replacements and temporary fracture fixation devices. Permanent orthopedic implants include the hip, knee, ankle, shoulder, elbow, wrist, and finger joints, which are expected to serve in the human body throughout the life span of the patients. On the other hand, temporary orthopedic implants including plates, screws, pins, wires, and intramedullary nails are needed to fix broken or fractured bones and are supposed to serve for a relatively short time just long enough to let bones heal. Since orthopedic implants must function under different working conditions in vivo, a good understanding of the fundamental requirements of orthopedic materials and subsequent biological response is crucial to the design and optimization of implants under physiological conditions in the human body. Selection of the proper materials for orthopedic implant relies on the specific applications. Metallic alloys, ceramics, and polymers are commonly used in orthopedic implants. These materials possess different physical, chemical, and biological properties that cater to specific applications. Despite the success of traditional materials, new and better biomaterials are being developed continuously to satisfy the ever-increasing demand.

OBJECTIVE: Endometriosis (endo) is an important gynaecological disease defined by the presence of ectopic endometrium (EM) outside the uterus and associated with pelvic pain and infertility. In this study we evaluated for the first time the feasibility of EM TRIzol extraction to obtain both a protein and mRNA fraction of sufficient quality from the same EM sample in order to perform combined proteomic analysis and mRNA microarray.DESIGN: Proteomic analysis of TRIzol-extracted eutopic endometrium.MATERIALS AND METHODS: Endometrial tissues were obtained during the late luteal phase (day 23-26) from 16 patients including controls with a normal pelvis (n=5) and patients with endometriosis (n=11) including minimal-mild (n=5) and moderate-severe (n=6) disease according to ASRM classification. We used Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF-MS): all samples were measured in duplicates on CM10 and IMAC30 protein chips in low molecular weight (CHCA) and high molecular weight (SPA). All mass peaks were used as input for a Least Squares Support Vector Machine (LS-SVM). Next, Leave one out cross validation (LOO-CV) was used to estimate independent test set performance.RESULTS: Profiling of endometrial samples by using different ProteinChip surfaces showed differential expression of proteins and peptides in the range between 7 - 17 kDa depending on chip and matrix type. The LOO-CV performance of the LS-SVM models indicated an Area under the ROC curve (AUC) between 85%-100% (depending on chip and matrix type) for the diagnosis of endometriosis. These results will be discussed in relationship to the mRNA microarray results obtained on the same EM samples (Sherwin et al, 2008).CONCLUSIONS: Protein profiling using ProteinChip array after TRIzol preparation is feasible and reproducible and allows combined proteomic analysis and mRNA microarray on the same EM sample. OBJECTIVE: Endometriosis (endo) is an important gynaecological disease defined by the presence of ectopic endometrium (EM) outside the uterus and associated with pelvic pain and infertility. In this study we evaluated for the first time the feasibility of EM TRIzol extraction to obtain both a protein and mRNA fraction of sufficient quality from the same EM sample in order to perform combined proteomic analysis and mRNA microarray. DESIGN: Proteomic analysis of TRIzol-extracted eutopic endometrium. MATERIALS AND METHODS: Endometrial tissues were obtained during the late luteal phase (day 23-26) from 16 patients including controls with a normal pelvis (n=5) and patients with endometriosis (n=11) including minimal-mild (n=5) and moderate-severe (n=6) disease according to ASRM classification. We used Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF-MS): all samples were measured in duplicates on CM10 and IMAC30 protein chips in low molecular weight (CHCA) and high molecular weight (SPA). All mass peaks were used as input for a Least Squares Support Vector Machine (LS-SVM). Next, Leave one out cross validation (LOO-CV) was used to estimate independent test set performance. RESULTS: Profiling of endometrial samples by using different ProteinChip surfaces showed differential expression of proteins and peptides in the range between 7 - 17 kDa depending on chip and matrix type. The LOO-CV performance of the LS-SVM models indicated an Area under the ROC curve (AUC) between 85%-100% (depending on chip and matrix type) for the diagnosis of endometriosis. These results will be discussed in relationship to the mRNA microarray results obtained on the same EM samples (Sherwin et al, 2008). CONCLUSIONS: Protein profiling using ProteinChip array after TRIzol preparation is feasible and reproducible and allows combined proteomic analysis and mRNA microarray on the same EM sample.

BACKGROUND Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. METHODS This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). RESULTS Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. CONCLUSIONS Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity.

OBJECTIVE: Endometriosis is a benign gynaecological condition, associated with pelvic pain and infertility.The lack of a reliable and specific method for the early detection of endometriosis often results in delayed diagnosis. Recent studies have shown that the complement system can be involved in the pathogenesis of endometriosis.The aim of this study was to test the hypothesis that the plasma concentration of complement factor C3a (anaphylatoxin) can be used as a non-invasive test in the diagnosis of endometriosis. DESIGN: Patients with endometriosis were compared with controls. MATERIALS AND METHODS: Plasma was collected during menstruation (n=49), the follicular (n=55) and the luteal phase (n=56) in 160 patients with (n=109) or without (n=51) laparoscopically and histologically confirmed endometriosis. Endometriosis was staged according to the American Society of Reproductive Medicine (ASRM) in minimal (n=27), mild (n=27), moderate (n=26) or severe (n=29) endometriosis. The C3a-des-Arg concentration in plasma was measured by Elisa according to the instructions of the manufacturer (Quidel, San Diego, USA). The sensitivity was 34ng/ml and the Intra assay coefficient of variation ranged from 1.5, 2.8% and Inter assay coefficient of variation ranged from 11, 23%. All data are presented as mean +/− SD and were analyzed using t-tests and ANOVA with Dunn's Multiple Comparison test. Power calculation for this experiment was 90%. RESULTS: The plasma C3a-des-Arg concentration was comparable between patients with (123±103ng/ml) and without (128±112ng/ml) endometriosis & was also comparable between patients with minimal (123±54ng/ml), mild (119±49ng/ml), moderate (97±43ng/ml) and severe (152±182ng/ml) endometriosis. The Dunn's Multiple Comparison test revealed no significant difference between control and diseased (control and stage I-II, control and stage III-IV and between stage I-II and stage III-IV).The plasma C3a-des-Arg concentration was also comparable in women with and without endometriosis during menstruation (110±86ng/ml and 123±69ng/ml, respectively), during the follicular phase (111±49ng/ml and 146±159 ng/ml, respectively) & during the luteal phase (146±146ng/ml and 116±92ng/ml, respectively). CONCLUSIONS: In this study, we did not confirm our hypothesis that plasma C3a levels can be used as diagnostic test for endometriosis.

BACKGROUNDEndometriosis occurs in 10% of women and is currently diagnosed by invasive laparoscopic testing. We tested the hypothesis that endometrial gene expression in late secretory phase endometrium differs between patients with and without endometriosis.METHODSTen patients with laparoscopically proven endometriosis (minimal/mild n = 5 and moderate/severe n = 5) and six controls, underwent endometrial biopsy in the late secretory phase (Day 23 onwards). Microarray interrogation of eutopic endometrial gene expression was performed.RESULTSMicroarray data were obtained for all control samples and eight samples from the endometriosis patients (n = 4 minimal/mild, n = 4 moderate/severe disease). Eight genes were identified as up-regulated and one gene was down-regulated in all endometriotic samples (more than 1.75-fold, P < 0.01). Real-time PCR analysis of protocadherin-17 (PCDH17), protein tyrosine phosphatase, receptor type, R (PTPRR) and interleukin-6 signal transducer (IL6ST) expression validated the microarray findings.CONCLUSIONSExpression of very few transcripts differs, in late secretory eutopic endometrium, between controls and patients with endometriosis. The median fold changes of these genes are small. No transcripts were identified that could discriminate between minimal/mild and moderate/severe endometriosis. Therefore, interrogation of the late secretory endometrial transcriptome is not likely to form the basis of a minimally invasive diagnostic test for endometriosis.

Endometriosis is more frequently diagnosed in patients with infertility than in a normal population. The goal of this paper is to give an overview of the clinical and fundamental evidence for a possible link between endometriosis and (recurrent) miscarriage or implantation failure after treatment with assisted reproductive technology. According to the literature, there is insufficient evidence for an association between endometriosis and (recurrent) miscarriage, but there is, however, epidemiological evidence to support the link between endometriosis and recurrent implantation failure after assisted reproduction. This can possibly be explained by alterations in humoral and cell-mediated immunity in women with endometriosis. Humoral immunological changes include increased formation of antibodies against endometrial antigens, anti-laminin-1 auto-antibodies and other auto-immune antibodies (e.g. antiphospholipid). Cell-mediated immunological changes include alterations in peritoneal and follicular fluid immune cells and cytokines. The possible negative effect of these immunological changes on folliculogenesis, ovulation, oocyte quality, early embryonic development and implantation in women with endometriosis suggests that infertility in endometriosis patients may be related to alterations within the follicle or oocyte, resulting in embryos with decreased ability to implant.

tion has oft en been criticized, there is currently no better method available. Th e stage of endometriosis is positively correlated with the degree of subfertility, but not or not as clearly with the degree of pelvic pain [4–6] . Surgical excision of endometriosis, even in mild stage disease, is an eff ective treatment for both endometriosis-associated subfertility and pain. Th e spontaneous pregnancy rate following surgery is negatively correlated with the degree of endometriosis [4] . Th e degree of pain relief following surgery is more pronounced in women with mild to severe endometriosis than in women with minimal endometriosis [7–9] . Current medical therapy of endometriosis is based on hormonal suppression and is eff ective for pain but useless for endometriosis-associated subfertility [5] . Since most women with endometriosis are of reproductive age, the active or passive desire to become pregnant later in life is an important issue. Early diagnosis of endometriosis in women who try to conceive should enable gynecologists to detect and excise endometriosis before the disease has progressed to a moderate to severe stage, in order to preserve or improve fertility as much as possible. In the presence of subfertility with a history of cyclic or chronic pelvic pain, combined with a clinical examination that is positive for pain, and an ultrasound positive for ovarian endometriotic cysts or deep endometriotic nodules, the probability of endometriosis is so high Endometriosis is defi ned as the presence of endometrial-like tissue outside the uterus, is associated with a chronic infl ammatory reaction in the pelvis and oft en results in subfertility and pain. Endometriosis occurs mainly in women of reproductive age (16–50 years), is estrogen-sensitive, and has a progressive character in at least 50%, but the rate and risk factors for progression are unpredictable and unknown, respectively. Th e diagnosis of endometriosis can be suspected in women with a history of dysmenorrhea, deep dyspareunia, chronic pelvic pain with or without subfertility, although it is possible that endometriosis is completely asymptomatic. In speculo inspection of cervix and vagina may show a small cervical diameter [1] , lateral cervical displacement [2] and rarely blue discoloration suggestive for cervical or vaginal endometriosis. Clinical abdominal and bimanual pelvic examination, preferably during the menstrual period, may reveal a painful lower abdomen, painful adnexal masses, painful uterosacral ligaments, and painful mobilization of the uterus. Vaginal ultrasound is an adequate diagnostic method to detect ovarian endometriotic cysts and deeply infi ltrative endometriotic noduli, but does not rule out peritoneal endometriosis or endometriosis-associated adhesions. Endometriosis is classifi ed in four stages: minimal, mild, moderate and severe [3] . Although this classifi caPublished online: May 4, 2006

BACKGROUNDInflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha), are important in the pathogenesis of endometriosis. We assessed the efficacy of anti-TNF monoclonal antibody (mAb, c5N), known to prevent induced endometriosis in baboons, in reducing established endometriosis in baboons.METHODSThis prospective, randomized, blinded, controlled study was conducted in baboons at the Institute of Primate Research (IPR), Nairobi, Kenya. Endometriosis was induced in 18 adult female baboons (Papio anubis) with regular menstrual cycles and a normal pelvis; the extent of endometriosis was documented by videolaparoscopy 25 days later. The baboons were then randomly assigned to receive a single infusion of either placebo (n=7, 5 ml/kg) or c5N (n=11, 5 mg/kg). Follow-up laparoscopy was performed 25 days later to document any differences in the number, surface area and estimated volume of lesions between the two groups and between the first and the second laparoscopies in each group. Representative biopsies of at least one endometriotic lesion per baboon were obtained at the final laparoscopy.RESULTSSignificant reductions in total surface area, estimated total volume of endometriotic lesions and both number and surface area of red lesions were observed after treatment with c5N, but not after placebo treatment, when compared to the initial laparoscopy. Conversely, a significant increase in the number of typical and red lesions was observed after placebo treatment when compared to the initial laparoscopy. Neither c5N nor placebo treatment affected the menstrual cycle.CONCLUSIONIn baboons with induced endometriosis, anti-TNF-mAb (c5N) treatment significantly reduced the extent of endometriosis, mainly due to reducing both the number and surface area of red lesions. These findings suggest that anti-TNF-mAb therapy may have therapeutic potential for active peritoneal endometriosis.

Tumor specific peptides recognized by T lymphocytes infiltrating solid tumors, as well as the corresponding T cell receptor (TcR) repertoire usage, have been extensively investigated. By contrast, tumor infiltrating B cells and their immunoglobulin (Ig) repertoire have been studied only in a limited number of tumors. The objective of the present study was to determine, whether DNA sequence analysis of the expressed immunoglobulin variable regions of B cells that infiltrate breast cancer, could be used to reveal a potential specific tumor binding capacity of the antibodies. To answer this question, about 200 expressed Ig heavy (VH) and light chain variable gene (VL) regions were cloned, sequenced and comparatively analysed from a typical medullary beast carcinoma (MBC), where the massive B and plasma cell infiltration correlates with favourable prognosis despite of its high grade. The tumor infiltrating B cell Ig heavy and light chain sequences could be classified into clusters, families and subgroups, based on the identity level to germline, showing a pattern of oligoclonality. Some overrepresented clusters could be determined. In the course of a detailed analysis and search in Blastn database, a number of VH and VL sequences showed more than 99% homology to DNA sequences of Ig VH region, with proved tumor antigen binding capacity. Our data suggest, that potential tumor binder Ig VH and VL sequences might be selected using a detailed immunoglobulin variable region analysis. This new approach might have a benefit for further antibody engineering, as difficulties in search for tumor binders by phage library selection might be reduced and the time for selection shortened.

variable region genes was performed.A single chain Fv phage display mini-library was generated from the selected VH and VL genes.