Fibromuscular differentiation in deeply infiltrating endometriosis is a reaction of resident fibroblasts to the presence of ectopic endometrium.

BACKGROUND: In this study, we characterized the fibromuscular (FM) tissue, typical of deeply infiltrating endometriosis, investigated which cells are responsible for the FM reaction and evaluated whether transforming growth factor-beta (TGF-beta) signaling is involved in this process. METHODS: FM differentiation and TGF-beta signaling were assessed in deeply infiltrating endometriosis lesions (n = 20) and a nude mouse model of endometriosis 1, 2, 3 and 4 weeks post-transplantation. The FM reaction was evaluated by immunohistochemistry using different markers of FM and smooth muscle cell differentiation (vimentin, desmin, alpha-smooth muscle actin, smooth muscle myosin heavy chain). TGF-beta signaling was assessed by immunostaining for its receptors and phosphorylated Smad. RESULTS: Deeply infiltrating endometriosis lesions contain myofibroblast-like cells that express multiple markers of FM differentiation. Expression of TGF-beta receptors and phospho-Smad was more pronounced in the endometrial component of the lesions than in the FM component. In the nude mouse model, alpha-smooth muscle actin expression was observed in murine fibroblasts surrounding the lesion, but not in human endometrial stroma. CONCLUSIONS: FM differentiation in deeply infiltrating endometriosis is the result of a reaction of the local environment to the presence of ectopic endometrium. It shares characteristics with pathological wound healing, but cannot be explained by TGF-beta signaling alone.

Transforming growth factor beta1 gene polymorphism 509C/T in deep infiltrating endometriosis.

Deep infiltrating endometriosis is characterized by the presence of nodular lesions largely composed of fibromuscular tissue. Transforming growth factor beta 1 (TGF-beta1) is the cytokine most causatively associated with disorders characterized by fibrosis throughout the body. Therefore, the hypothesis was tested that mechanisms increasing the fraction of biologically active TGF-beta1, such as TGF-beta 1 gene polymorphisms, lead to an increased risk of developing deep infiltrating endometriosis. The frequency of the -509C/T polymorphism of the TGF-beta 1 gene was tested in women with deep infiltrating endometriosis (n = 72), gynecological patients without symptoms of endometriosis (n = 95) and healthy females (n = 93). Detection of the -509C/T polymorphisms was performed using PCR-restriction fragment length polymorphism analysis. We did not observe statistically significant differences in the frequency of the -509C/T polymorphism between the groups. Our study does not support an association between the -509C/T polymorphism of the TGF-beta 1 gene and an increased risk of deep infiltrating endometriosis.

Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human.

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

Hypoxia contributes to development of recurrent endometrial carcinoma.

Tumor hypoxia can trigger the induction of angiogenesis. High microvessel density (MVD) as well as hypoxia-inducible factor-1alpha (HIF-1alpha) have been related to recurrent disease and tumor aggressiveness, respectively. In this study, MVD and hypoxic status were investigated in primary and recurrent endometrial carcinomas. A total of 65 primary tumors of patients with recurrent endometrial carcinoma (n = 40), and without recurrent endometrial carcinoma (n = 25) were studied. Immunohistochemical analysis was performed on paraffin-embedded tumor tissue. MVD was determined by quantitative analysis of CD31/FVIII positive vessels. Tumor hypoxia was estimated by evaluating the expression of the hypoxia-regulated gene HIF-1alphaand its target gene carbonic anhydrase IX (CA-IX). An additional 23 recurrent tumors were available for determination of MVD and HIF-1alpha expression. Effects of hypoxia on tumor protein p53 (TP53) expression were evaluated in the endometrial cancer cell lines (ECC-1), Ishikawa (derived from adenocarcinomas), and AN3CA (derived from a lymph node metastasis). MVD, CA-IX, and HIF-1alpha expression were not significantly different in primary tumors of patients with recurrence compared to the control tumors. The MVD was significantly lower, and HIF-1alpha expression was significantly higher in recurrent tumors when compared with their primary tumors (paired t test, P < 0.05). HIF-1alpha expression correlated well with TP53 expression levels in primary tumors, but not in recurrences. TP53 protein levels were highest in AN3CA cells. Hypoxic conditions induced TP53 protein in ECC-1 and Ishikawa, but not AN3CA cells. We conclude that MVD, CA-IX, and HIF-1alpha expression are not independent prognostic markers for recurrent endometrial carcinoma. The low MVD, increased HIF-1alpha protein levels, dissociation of hypoxia, and TP53 protein induction in the metastatic tumor cells (AN3CA) support a role for hypoxia in the development of recurrent endometrial carcinoma.

Progesterone receptor polymorphism +331G/A is associated with a decreased risk of deep infiltrating endometriosis.

BACKGROUND: Alterations in the progesterone receptor (PR) are considered a risk factor for the development of endometriosis. In this study, the frequencies of the PROGINS and +331G/A polymorphisms of the PR gene were determined in deep infiltrating endometriosis and correlated with the expression of the PR protein. METHODS AND RESULTS: The frequencies of the PR polymorphisms were determined in women with deep infiltrating endometriosis (n = 72), women with adenomyosis in the uterine wall (n = 40), gynaecological patients without symptomatic endometriosis (n = 102) and healthy females (n = 93). Detection of +331G/A and PROGINS polymorphisms was performed using PCR-restriction fragment length polymorphism (RFLP) analysis. Expression of PR-A and PR-B protein was assessed with immunohistochemistry. The allelic frequency of the polymorphic allele +331A was lower in women with endometriosis (P < 0.01) and adenomyosis (P < 0.02) compared with healthy females. The frequency of the PROGINS polymorphism did not differ between the groups. The mean staining index (SI) for PR-B in endometriotic epithelium was higher in the presence of the +331A polymorphic allele (n = 2) (P < 0.001) compared with +331G/G individuals (n = 61). The PROGINS polymorphism did not affect the SI for PR-A and PR-B. CONCLUSIONS: The presence of the PR gene polymorphic allele +331A is associated with a reduced risk of deep infiltrating endometriosis and adenomyosis compared with healthy population controls. The PROGINS polymorphism does not seem to modify the risk of deep infiltrating endometriosis.

RASSF1A methylation and K-ras and B-raf mutations and recurrent endometrial cancer.

BACKGROUND: Aberrations in mediators of Ras signaling may increase the risk of developing recurrent endometrial carcinoma. PATIENTS AND METHODS: Primary tumors of patients with (n = 44) and without (n = 44) recurrent stage I endometrioid endometrial carcinoma were compared regarding the presence of K-ras mutations (codons 12 and 13), B-raf mutations (V599), and RASSF1A gene promoter methylation. RESULTS: K-ras mutations were present in 18% of the patients independent of recurrent disease. No B-raf mutations were found. RASSF1A methylation was demonstrated in 85% of endometrial carcinomas, independent of recurrence. The presence of K-ras mutations and RASSF1A promoter methylation were not related, either directly or inversely. Analysis in premenopausal endometrial carcinomas demonstrated K-ras mutations in 40%, no B-raf mutations, and RASSF1A promoter methylation in 70% of the cases. RASSF1A methylation was also observed in samples of cyclic (n = 14), hyperplastic (n = 8), and atrophic (n = 13) endometrial tissues in 21%, 50% and 38%, respectively. CONCLUSIONS: RASSF1A methylation was observed in a high frequency in endometrioid endometrial carcinoma whereas K-ras and B-raf mutations were observed in a low frequency. No association was observed with the development of recurrent disease. High-frequency RASSF1A methylation in premenopausal carcinomas and an increased frequency in endometrial hyperplasia indicate that this may be an early event in endometrial carcinogenesis.

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism.

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium.

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.

Vascular development in endometriosis.

Endometriosis, defined as the presence of endometrial tissue outside the uterus, is an estrogen-dependent disease which causes pelvic pain and subfertility in women of reproductive age. The condition has a dramatic impact on the professional, social and marital life of sufferers. Direct and indirect evidence suggests that angiogenesis is required for the development and persistence of endometriosis. In this review the state-of-the-art with regard to our understanding of the role of angiogenesis in the ectopic implantation and survival of menstrual endometrial tissue will be discussed.

Aberrations in the progesterone receptor gene and the risk of recurrent endometrial carcinoma.

A case-control study was performed in order to determine whether expression of the progesterone receptor (PR) and/or aberrations of the PR gene contribute to the development of recurrent endometrial carcinoma. Primary tumours from 44 patients with recurrence of stage I endometrial carcinoma (patients) within 3 years after initial treatment were compared with tumours from 44 matched patients who were free of recurrence for a minimum of 3 years (controls). Paraffin wax-embedded primary tumours (n = 88) and recurrent tumours (n = 32) were analysed immunohistochemically for PR expression. A staining index (SI = 0-9) based on the staining intensity and the number of stained cells was calculated. DNA extracted from paraffin wax-embedded tissues was subjected to PCR-restriction fragment length polymorphism analysis (PCR-RFLP) for determination of the PROGINS DNA sequence alterations and the +331G/A-promoter polymorphism. Low PR expression (SI < 1.0) was observed in 7% of primary tumours derived from controls, 25% of primary tumours from patients with recurrence, and 38% of recurrent tumours. The expression of PR was significantly lower in primary tumours from patients with recurrence (SI = 4.0 +/- 0.5) than in the tumours in the control group (SI = 5.6 +/- 0.5) (T-test for paired analysis, p < 0.05). The PROGINS and +331G/A-promoter polymorphism were not related to age at diagnosis, tumour grade or myometrial invasion. The +331G/A-promoter polymorphism was present in 14% of primary tumours from patients without recurrence, compared with 17% of patients with recurrence. The PROGINS polymorphism was observed in 16% of primary tumours from patients without, and in 34% of patients with, recurrence (OR 2.6; 95% CI: 0.9-7.6). Most interestingly, patients who carried the PROGINS variant and in whom a PR-expressing tumour was diagnosed were at significantly enhanced risk of relapse (OR 4.7; 95% CI: 1.3-17.1). In conclusion, low PR expression tended to be associated with recurrent disease, and PR expression in tumours from patients carrying the PROGINS allele was predictive of the risk of recurrence.

APC, beta-catenin, and E-cadherin and the development of recurrent endometrial carcinoma.

Endometrial carcinoma, generally, has a good prognosis. However, in some patients, the tumor appears to behave very aggressively, a course that cannot be explained with histopathological characteristics. More insight into the molecular background can be valuable to clarify these differences in tumor behavior. The three components associated with the Wnt pathway--i.e., adenomatous polyposis coli (APC), beta-catenin, and E-cadherin--were evaluated in a case-control study of 28 patients with stage-I endometrial carcinomas to determine their involvement in the development of recurrent disease. Mutation analysis of the mutation cluster region of the APC gene, determination of gene promoter methylation status of the APC-1A and E-cadherin genes, and immunohistochemical analysis of APC, E-cadherin, and beta-catenin were performed using paraffin-embedded tumor tissue. Twenty-one APC gene mutations were detected in 12 of 28 (43%) patients. Only three mutations would result in a stopcodon in the APC gene. APC gene promoter methylation was assessed in 12 of 28 (43%) patients. APC immunostaining was absent in two of 24 (8.3%) patients. The occurrence of APC mutations, APC gene promoter methylation, and APC immunostaining were not predictive for recurrence. No E-cadherin expression was observed in four of 24 patients (17%). E-cadherin gene promoter methylation could not be detected in any of the patients. The absence of E-cadherin expression was predictive for distant metastases, but not for local recurrence. Nuclear localization of beta-catenin was present in nine of 24 (38%) patients and was not predictive for recurrent disease. Involvement of epigenetic and genetic aberrations in APC and beta-catenin genes seems to be of minor importance for the development of local recurrences and distant metastases. Although the number of patients is limited, E-cadherin expression appears to be predictive for the development of distant metastases in endometrial carcinoma.

Defective mismatch repair and the development of recurrent endometrial carcinoma.

OBJECTIVE: To investigate whether defective DNA mismatch repair (MMR) defines a subgroup at risk for recurrence in sporadic endometrial carcinoma patients. METHODS: Primary tumors from 44 patients with recurrent stage I endometrial carcinoma were compared after matching, with tumors of 44 patients being free of recurrence for minimal 3 years. Paraffin-embedded primary tumors (n = 88) and recurrent tumors (n = 32) were subjected to immunohistochemical analysis for hMSH2 and hMLH1 expression. Subsequently, a staining index (SI = 0-9) was calculated based on staining intensity and quantity. DNA was extracted from paraffin-embedded tissues, and promoter methylation of hMLH1 was determined by nested methylation-specific PCR (MSP). Microsatellite instability (MSI) was assessed by BAT-26 or BAT-25. RESULTS: Low hMSH2 expression was observed in 2% of primary tumors of control patients without recurrence, 14% of primary tumors of patients with recurrence, and 0% of recurrent tumors. Low hMLH1 expression was observed in 32%, 19%, and 22%, respectively. hMLH1 gene promoter methylation was detected in 50%, 47%, and 32%, and MSI was found in 16%, 14%, and 30%, respectively. No significant differences were found between primary tumors of patients with and without recurrence with respect to hMSH2 and hMLH1 expression, hMLH1 promoter methylation, and MSI. When primary and recurrent tumors were compared, there was an increased correlation of hMLH1 methylation with low hMLH1 expression and MSI in recurrent tumors. CONCLUSION: MSI, hMLH1 promoter methylation, and the expression of hMLH1 and hMSH2 are not predictive for the development of recurrent stage I endometrial carcinoma. In the progression of tumor, "de novo" hMLH1 methylation rarely occurs, instead there is further derailment of the MMR pathway in affected tumors.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells.

BACKGROUND: Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). METHODS: Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. RESULTS: Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and alpha-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. CONCLUSIONS: In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Retinol and estradiol regulation of retinol binding protein and prostaglandin production by porcine uterine epithelial cells in vitro.

Secretion into the uterine lumen follows a precise pattern during early pregnancy. Near the end of the second week of pregnancy and coincident with elongation of conceptuses, retinol, retinol binding protein (RBP), estradiol (E2), and prostaglandins E (PGE) and F (PGF) increase in the uterine lumen, and RBP mRNA increases in the endometrium. In the present studies the potential for E2 (0.1 microM) and retinol (10 microM) to regulate RBP and PG production by cultured luminal (LEC) and glandular (GEC) epithelial cells collected from postpubertal females and LEC from prepubertal gilts was examined. Endometrial tissue was collected surgically from cyclic and pregnant females (n = 8) on d 10 and 13 postestrus (first day of estrus = d 0) and from 120- and 150-d-old prepubertal gilts that were treated with progesterone (P4) (2.2 mg x kg(-1) x d(-1), n = 6) or corn oil (n = 6) for 14 d prior to tissue collection. The LEC from postpubertal females responded to retinol with increased (P < 0.05) RBP, PGE, and PGF in culture medium and increased (P < 0.07) RBP mRNA but E2 decreased (P < 0.05) RBP and RBP mRNA and had no effect on prostaglandins. No E2 or retinol effects on secretions of GEC occurred in vitro, but a day x pregnancy status interaction (P < 0.06) affected PGE output by the GEC. Secretion of PGE was greater when GEC were collected on d 10 of pregnancy than from d-10 cyclic or d-13 pregnant or cyclic females. Both E2 and retinol stimulated (P < 0.05) secretion of RBP by LEC isolated from prepubertal gilts, but their effects were not additive. In vivo treatment of prepubertal gilts with P4 increased (P < 0.05) RBP and decreased (P < 0.05) PG production by LEC in vitro. Therefore responses to E2 and retinol differ between pre- and post-pubertal females, and retinol may function in the regulation of endometrial RBP and PG secretion.

Endometrial angiogenesis throughout the human menstrual cycle.

BACKGROUND: The timing and mechanisms of new blood vessel formation in the endometrium during the menstrual cycle are still largely unknown. In the present study we used the chick embryo chorioallantoic membrane (CAM) as an in-vivo assay for angiogenesis to assess the angiogenic potential of endometrium obtained at different stages of the menstrual cycle. METHODS: Endometrial fragments were explanted onto the CAM and, after 4 days of incubation, slides of the treated area were taken in ovo through a microscope for computerized image analysis. The vascular density index (VDI), a stereological estimate of vessel number and length, was obtained by counting the intersections of vessels with five concentric circles of a circular grid superimposed on the computerized image. RESULTS: We demonstrated that human endometrium has angiogenic potential throughout the menstrual cycle. Furthermore, there was a significant difference in angiogenic response between the stages of the menstrual cycle (P = 0.01). The VDIs of the early proliferative, early and late secretory stage were significantly higher than the VDI of the late proliferative phase. CONCLUSIONS: Elongation of existing vessels during the early proliferative phase as well as growth and coiling of the spiral vessels during the secretory phase may demand far higher angiogenic activity than outgrowth and maintenance of vessels during the late proliferative phase.

The Mesothelium, Teflon or Velcro? Mesothelium in endometriosis pathogenesis.

Sampson's transplantation theory for the pathogenesis of peritoneal endometriosis is widely accepted. The events that take place, however, on the cellular and subcellular level during the transition of endometrial tissue in the abdominal cavity into peritoneal endometriosis remain controversial. The mesothelium plays a central role in the debate on this subject. The interaction between endometrium and peritoneum has been studied in an in-vitro model using amnion, peritoneum and mesothelial cells in culture on the one hand and cyclic and menstrual endometrium on the other hand. The results of these studies indicate that (i) an intact mesothelial lining prevents adhesion of shed endometrial tissue, (ii) shed endometrial tissue adheres to the peritoneal extracellular matrix and (iii) menstrual effluent creates its own adhesion sites by damaging the mesothelial lining thus exposing the extracellular matrix. Therefore we conclude that the mesothelium has the properties of Teflon, while the extracellular matrix resembles Velcro.

Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Menstruum induces changes in mesothelial cell morphology.

In previous studies, we have shown that menstrual endometrium preferentially adheres to the subepithelial lining of the peritoneum. It remains to be elucidated, however, whether this damage is preexisting or inflicted by the menstrual tissue itself. We hypothesized that the menstrual tissue itself damages the peritoneum. To investigate this, the viability of menstrual endometrial tissue in peritoneal fluid (PF) was evaluated and the morphologic changes in the mesothelial cells were studied by in vitro cocultures of menstruum with mesothelial cell monolayers. Menstruum was collected with a menstrual cup. Endometrial tissue was isolated from the menstruum, resuspended in culture medium or in the cell-free fraction of PF and cultured for 24, 48 or 72 h. A 3(4, 5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to obtain a relative measure of viable adhered endometrial cells. Mesothelial cells isolated from human omental tissue were cultured on Matrigel or uncoated plastic. At confluence, overnight cocultures were performed and scanning electron microscopy was used to evaluate the morphologic changes. The viability of endometrial fragments was 84% (n = 36, p < 0.05), 82% (n = 27, not significant) and 104% (n = 14, not significant) when cultured in the cell-free fraction of PF for 24, 48 and 72 h, respectively, when compared to medium with 10% fetal calf serum. Menstrual endometrial fragments or menstrual serum added to and cocultured with mesothelial cells induced severe morphologic alterations of the latter, including retraction, shrinking and gap formation. Similar morphologic changes were observed when mesothelial cells were cocultured with menstrual endometrial fragments in PF or in culture inserts. Incubation with conditioned medium from cultured menstrual endometrium induced similar but less pronounced changes in morphology. In conclusion, menstrual endometrial fragments remain viable in PF in vitro for at least 72 h. Antegradely shed menstruum induces changes in mesothelial cell morphology, including retraction and shrinking with exposure of the underlying surface. These findings suggest that menstruum is harmful to the peritoneal lining. Therefore, by local destruction of the mesothelial layer, menstrual endometrium is able to create sites for adhesion.