TRIzol treatment of secretory phase endometrium allows combined proteomic and mRNA microarray analysis of the same sample in women with and without endometriosis.

BACKGROUND: According to mRNA microarray, proteomics and other studies, biological abnormalities of eutopic endometrium (EM) are involved in the pathogenesis of endometriosis, but the relationship between mRNA and protein expression in EM is not clear. We tested for the first time the hypothesis that EM TRIzol extraction allows proteomic Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF MS) analysis and that these proteomic data can be related to mRNA (microarray) data obtained from the same EM sample from women with and without endometriosis. METHODS: Proteomic analysis was performed using SELDI-TOF-MS of TRIzol-extracted EM obtained during secretory phase from patients without endometriosis (n = 6), patients with minimal-mild (n = 5) and with moderate-severe endometriosis (n = 5), classified according to the system of the American Society of Reproductive Medicine. Proteomic data were compared to mRNA microarray data obtained from the same EM samples. RESULTS: In our SELDI-TOF MS study 32 peaks were differentially expressed in endometrium of all women with endometriosis (stages I-IV) compared with all controls during the secretory phase. Comparison of proteomic results with those from microarray revealed no corresponding genes/proteins. CONCLUSION: TRIzol treatment of secretory phase EM allows combined proteomic and mRNA microarray analysis of the same sample, but comparison between proteomic and microarray data was not evident, probably due to post-translational modifications.

Increased exposure to dioxin-like compounds is associated with endometriosis in a case-control study in women.

Although endometriosis is thought to be an environmental disorder initiated by dioxin exposure, this association is controversial. This study was performed to test the hypothesis that endometriosis occurs more often in women exposed to higher concentrations of dioxin-like compounds (DLC) than in those women exposed to lower concentrations. Plasma samples collected prior to laparoscopic surgery from 96 women with endometriosis and 106 control patients with a normal pelvis were measured for DLC concentrations using the dioxin-responsive chemical-activated luciferase expression bioassay. The results showed that concentration (mean+/-SD) of DLC was marginally higher in patients with endometriosis (22.3+/-9.3pg CALUX-TEQ/g lipid) than in controls (20.5+/-10.8pg). After categorization of patients in a group with 'low' plasma concentrations (<25th centile) and a group with 'high' plasma concentrations (>75th centile) of DLC, the age-adjusted odds ratio to have endometriosis was 2.44 (95% CI 1.04-5.70; P=0.04) for women with high concentrations of DLC and it increased to 3.01 (95% CI 1.06-9.04; P=0.03) when only women with moderate severe endometriosis were considered. In conclusion, women exposed to higher plasma concentrations of DLC were at higher risk of having endometriosis than women exposed to lower concentrations of DLC within normal environmental concentrations.

Reassessing the evidence for the link between dioxin and endometriosis: from molecular biology to clinical epidemiology.

A 1993 study reporting the link between exposure to dioxin and the risk of developing endometriosis in rhesus monkeys prompted many investigators to look suspiciously at dioxin. Since 1993, many in vitro, animal and epidemiological studies have been published, but the link between dioxin exposure and endometriosis is still unclear. The aim of our review is to present a summary of the biological effects of dioxin and its aryl hydrocarbon receptor, and to reassess the evidence presented in published, in vitro, preclinical and epidemiological studies regarding the association between dioxins and endometriosis. Although in vitro and animal studies provide results in support for a role of dioxins in the pathogenesis of endometriosis, caution should be exercised since these findings are mostly context dependent and since negative findings from these studies are rarely published. On the basis of our review of original epidemiological studies, no significant evidence can be found to support a link between dioxins and endometriosis in women. This observation can be explained by positive publication bias and by significant methodological problems associated with these studies, or by the absence of such a link. In conclusion, it seems that there is insufficient evidence at this moment in support of the hypothesis that dioxin exposure may lead to increased risk of developing endometriosis in women.

[Environmental dioxin compounds as the cause of endometriosis and other diseases]. / A környezetszennyezo dioxinvegyületek mint az endometriosis és egyéb betegségek kórokai.

There has been much debate of late about whether or not dioxin, an industrial toxin, could be a causative agent in the onset of endometriosis, a gynaecological disease associated with infertility and pain. Studies found either no difference in serum dioxin concentrations when cases were compared to controls or a non-significant increase, or reached low statistical power. The introductory results on Rhesus monkey contradict with the observations on mice fed with dioxin and oestrogen simultaneously. Genetic comparison shows that human belongs to the dioxin resistant races so dioxin concentrations measured in the population could not cause disease especially not an oestrogen dependent one, like endometriosis.

Emerging drugs in endometriosis.

Endometriosis is a common, estrogen-dependent, gynaecological disease, defined as the presence of endometrial-like tissue outside the uterus. Although several medications are used for treatment of the disease, they are associated with high recurrence rates, considerable side effects and limited duration of application. Due to these limitations and to the impact of endometriosis on the quality of life of affected women, their environment and the society, there is a great need for new drugs able to abolish endometriosis and its symptoms. Studies in recent years investigating the (patho)physiological mechanisms involved in disease aetiology have fostered the development of novel therapeutic concepts for endometriosis, by targeting the hypothalamic-pituitary-gonadal axis, by selective modulation of estrogenic and progestogenic pathways, by inhibiting angiogenesis or by interfering with inflammatory and immunological factors. This article presents a brief summary of the currently available medications and an overview regarding the development of some of the most interesting and/or most promising novel drug candidates for endometriosis.

Tumor-infiltrating B cell immunoglobulin variable region gene usage in invasive ductal breast carcinoma.

A major focus of tumor immunology is to reveal the potential role and capacity of immunocompetent cells found in different solid tumor tissues. The most abundant infiltrating cells (TIL), the T lymphocytes have been investigated in details concerning T-cell receptor usage and specificity. However, B cells have hardly been investigated in this respect, although high cellular B-cell infiltration has been correlated with improved patients' survival in some breast carcinomas. This led to our objectives to study variable region gene usage of the tumor-infiltrating B cells in different breast carcinoma types. By defining the immunoglobulin repertoire of the tumor-infiltrating B lymphocytes in the most common invasive ductal carcinoma (IDC) of the breast we compared it to the rare medullary breast carcinoma (MBC). After phenotyping infiltrating ductal carcinomas, B cells were obtained from tumor tissue by microdissection technique. Numerous rearranged TIL-B immunoglobulin heavy chain V genes (VH) were amplified, cloned, sequenced, and comparatively analyzed. Some characteristics were found for both breast carcinoma types. The immunoglobulins produced by TIL-B in ductal carcinoma are highly matured and oligoclonal. We conclude that Ig variable region gene usage reveals similar and distinguishable characteristics of TIL-B immunoglobulin repertoires, which are representative of the nature of the immune responses in invasive ductal and medullary breast carcinomas.

Evaluation of endometrial biomarkers for semi-invasive diagnosis of endometriosis.

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.

Plasma C3a-des-Arg levels in women with and without endometriosis.

PROBLEM: The lack of a reliable method for early non-invasive detection of endometriosis often results in delayed diagnosis. 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. METHOD OF STUDY: The C3a concentration was analyzed using ELISA in 160 patients with (n = 109) or without (n = 51) endometriosis during menstruation (n = 49), follicular phase (n = 55), and luteal (n = 56) phase. RESULTS: Plasma C3a concentration was comparable between patients with [102 (27-2213) ng/mL] and without [105 (32-2340) ng/mL] (P = 0.84) endometriosis, also when assessed separately during menstruation, follicular phase, and luteal phase. CONCLUSION: We found no difference in C3a levels between women with and without endometriosis and did not confirm our hypothesis that plasma C3a levels can be used as diagnostic test for endometriosis.

Role of cytokines in the endometrial-peritoneal cross-talk and development of endometriosis.

A clear picture of the dynamic relationship between the endometrium and peritoneum is emerging as both tissues may participate in the spontaneous development of endometriosis. Various adhesion molecules, pro-inflammatory cytokines and chemoattractants cytokines have emerged as central coordinators of endometrial-peritoneal interactions. The peritoneal microenvironment which consists of the peritoneal fluid, normal peritoneum and peritoneal endometriotic lesions may play an active role in the pathogenesis of endometriosis, by harbouring most inflammatory responses that are triggered by the presence of endometrial cells, leading to recruitment of activated macrophages and leukocytes locally. Menstrual endometrium has the ability to bond and invade the peritoneal tissue. In baboons intrapelvic injection of menstrual endometrium permits the study of early endometrial-peritoneal interaction in an in vivo culture microenvironment and can lead to important insight in the early development of endometriotic lesions. In this review, we discuss the roles of the endometrial-peritoneal interactions, not only in disease development but also in the broader process of aetiopathogenesis.

Selective estrogen-receptor modulators and aromatase inhibitors: promising new medical therapies for endometriosis?

Endometriosis is an estrogen-dependent disease and estrogen-related pathways are imbalanced in women with endometriosis. One of the key enzymes in estrogen synthesis is aromatase. Inhibiting this pathway at several points is a promising idea for the treatment of endometriosis. The third generation of aromatase inhibitors is becoming more potent in efficacy, with fewer side effects than previous generations, but cotreatment with other hormones is needed to inhibit ovarian stimulation. Other components that promote estrogen synthesis such as COX-2 can also be potentially targeted. Selective estrogen-receptor modulators could also be interesting in view of their tissue-specific effect. However, all these new drugs are still in an early phase of development. At present, it is too early to conclude that aromatase inhibitors, COX-2 inhibitors or selective estrogen-receptor modulators really present any added value compared with the existing drugs that can be used to achieve hormonal suppression in the medical treatment of endometriosis.

Baboon model for the study of endometriosis.

Endometriosis is a benign, estrogen-dependent disease and is now recognized as an enigmatic disease owing to its various clinical manifestations and locations. The lack of a reliable and specific method for the early detection of endometriosis often results in delayed diagnosis. So far, research has born inadequate findings regarding understanding the basic etiology or pathophysiology of endometriosis. Animal models that accurately represent the cellular and molecular changes associated with the initiation and progression of human endometriosis have significant potential to facilitate the development of better methods for the early detection and treatment of endometriosis. A number of animal model systems have been developed for the study of this disease. These models replicate many of the known salient features of human endometriosis. This review provides an insight into the use of the baboon model for studies focused on understanding human endometriosis.

Future of endometriosis research.

In women of reproductive age, health economic costs are estimated to be considerably higher for endometriosis than for conditions such as Crohn's disease, migraine and hypertension, and similar to the cost of diabetes. However, more awareness of endometriosis among patients and politicians is needed to create a better climate for research funding in the area of endometriosis in particular, and women's health in general. Recent collaboration between patients, physicians and politicians in the EU has shown that such efforts can be successful. Many arguments exist to organize the clinical care for women with advanced endometriosis in centers of excellence, but continuing education of primary-care physicians also remains a priority. New molecular techniques are resulting in new hormonal and nonhormonal targets for the noninvasive diagnosis and treatment of endometriosis. A future diagnostic serum assay might contain various elements from inflammatory serum markers to genetic/microarray/proteomics markers, owing to the multifactorial features of endometriosis.

Endometriosis in African women.

Endometriosis is a gynecological disorder characterized by the growth of endometrial tissue outside the uterine cavity. Although the prevalence of endometriosis is well documented in women living in developed countries, studies on the prevalence of this disease among African women are still wanting. The current view is that endometriosis rarely affects women of African descent. However, in African-American women in the USA, endometriosis is one of the common indications for major gynecological surgery and hysterectomy and is associated with a long hospitalization and high hospital charges. Endometriosis may be more commonly found in infertile Caucasian or African-American women than in African-Indigenous women, but it is likely that the true prevalence of endometriosis in African-Indigenous women is under reported owing to inadequate facilities and demands of specialized skills for adequate assessment of the pelvis and recognition of the various types and appearances of the disease. Understanding the prevalence of endometriosis among African women will be instrumental in proper management of this disease in the African continent.

ProteinChip technology is a useful method in the pathogenesis and diagnosis of endometriosis: a preliminary study.

OBJECTIVE: To test the feasibility of ProteinChip (Ciphergen Biosystems, Inc., Fremont, CA) technology as a proteomic tool in discovering and identifying proteins that are differentially expressed in endometrium, endometriotic tissue, and normal peritoneum from women with and without endometriosis. DESIGN: Differential analysis of protein expression in women with and without endometriosis. SETTING: University hospital. PATIENT(S): A total of nine patients during their secretory phase (days 20-22) were selected for this study on the basis of cycle phase and presence/or absence of endometriosis. INTERVENTION(S): Twelve tissues used in the study included six endometrial biopsies from women with mild endometriosis (n = 3) and a normal pelvis (n = 3) as well as paired samples of peritoneal endometriotic lesions (n = 3) and macroscopically normal peritoneum biopsies (n = 3) from three women with endometriosis. MAIN OUTCOME MEASURE(S): Numerous expression differences were observed in the above comparisons, representing both up-regulation and down-regulation in protein and peptide expression levels. RESULT(S): Endometrial expression for a number of proteins and peptides in the range of 2.8-12.3 kDa was 3-24 times lower in women with endometriosis than in those without endometriosis. When compared with normal peritoneum, endometriotic lesions showed an increased expression for a set of proteins and peptides in the range of 3-96 kDa, and especially an up-regulated cluster of proteins between 22 and 23 kDa, identified to be transgelin, a smooth muscle actin-binding protein. CONCLUSION(S): This preliminary study demonstrated that differential protein profiling by using ProteinChip array technology is feasible, reproducible, and may be developed into a powerful tool for endometriosis research.

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.