Differential regulatory effect of progesterone on the proliferation and apoptosis of uterine leiomyoma tissue explants and primary leiomyoma cell cultures.

OBJECTIVE: The growth of uterine leiomyomas is regulated by progesterone, although the underlying molecular mechanisms are not fully understood. METHODS: Primary leiomyoma cells were isolated by standard method from 16 samples of uterine leiomyoma tissue. Uterine leiomyoma explants and primary leiomyoma cell cultures were exposed to progesterone in concentrations of 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml for 24 h. Cell apoptosis was assessed with Annexin V assays performed in cell cultures by flow cytometry. The expression of PR-A, PR-B, Ki67, Akt, ERK, PTEN and PPARγ mRNAs was estimated in cultured leiomyoma cells and tissue explants by real time RT-PCR. RESULTS: Treatment with progesterone promoted viability and proliferation of cultured leiomyoma cells in a dose-dependent manner. Low and high doses of progesterone decreased early apoptosis of leiomyoma cells. High concentrations of progesterone increased the number of living cells in Annexin V assays. High doses of progesterone increased the expression of Ki67 mRNA, while low doses increased the expression of PR-A mRNA in cultured leiomyoma cells and tissue explants. In cell cultures, low doses of progesterone increased the expression of PR-B mRNA and the expression of PTEN and PPARγ mRNAs in a dose-dependent manner. Exposure of leiomyoma tissue explants to progesterone led to increased expression of PR-B and ERK mRNAs in a dose-dependent manner. CONCLUSIONS: The effects of progesterone on the apoptosis and proliferation of leiomyoma cells was dose-dependent and different in cell cultures and leiomyoma explants, possibly as a result of impacts derived from the tumor microenvironment.

Changes of apoptosis regulation in the endometrium of infertile women with tubal factor and endometriosis undergoing in vitro fertilization treatment.

OBJECTIVE: To establish the relationship between the endometrial apoptosis parameters and endometrial receptivity in infertile women undergoing IVF treatment. METHODS: 73 women with tubal infertility, 27 infertile women with endometriosis and 13 healthy fertile women (control group) were recruited into the study. 53 women with tubal factor of infertility and 17 patients with endometriosis later entered in the IVF protocol. Samples of endometrial tissue were used as the material for investigation. Endometrium was collected on the 20-24 days of non-conceptual cycle, using the Pipell suction curette. XIAP, PTEN and HSP27 mRNAs expression in the endometrial tissue was assessed by real time RT-PCR. RESULTS: In women with tubal infertility the high level of XIAP, HSP27 and PTEN mRNAs expression in the endometrium was found. In infertile women with endometriosis the increase of XIAP and HSP27 mRNAs expression was noted. Success of the IVF outcome in women with tubal infertility was associated with maximal level of PTEN synthesis and in endometriosis group the pregnancy achievement after IVF treatment was noted in women with lower expression of XIAP mRNA. CONCLUSION: The high level of pro-apoptotic factors synthesis in the endometrium during window of implantation is associated with the readiness of endometrium to implantation.

Mechanisms regulating invasiveness and growth of endometriosis lesions in rat experimental model and in humans.

OBJECTIVE: To compare the expression of MMP-2, TIMP-2, and TGFbeta2 mRNA in experimental and human endometriotic lesions and to assess the possibility of its cytokine regulation. DESIGN: Experimental laboratory study. SETTING: Medical center. ANIMALS AND PATIENT(S): Thirty female Wistar rats, 17 women with endometriosis, 11 healthy women. INTERVENTION(S): Uterine transplants were attached to rat peritoneum via the surgical autotransplantation technique. The collection of endometriotic implants at 7, 14, and 21 days postsurgery and laparoscopic collection of peritoneal fluid, ectopic, and matched eutopic endometrium from women with endometriosis were performed. MAIN OUTCOME MEASURE(S): MMP-2, TIMP-2, TGFbeta2 mRNA expression in endometrium was assessed by real-time reverse-transcription polymerase chain reaction. RESULT(S): In rats, the increase of MMP-2 and decrease of TIMP-2 mRNA expression was noted at the 7th day, and an increase of TGFbeta2 mRNA expression was seen at the 14th day postsurgery. In humans, elevation of TIMP-2 mRNA expression in eutopic endometrium and of MMP-2, TGFbeta2 mRNA expression in ectopic endometrium was observed. Autologous peritoneal fluid stimulated MMP-2 mRNA expression in eutopic endometrium of women with endometriosis. Cytokines derived from ectopic lesions mononuclear cells increased TGFbeta2 mRNA expression in endometrium of healthy women. CONCLUSION(S): Supposedly MMP-TIMP balance is important in promoting endometriotic tissue invasion and TGFbeta2 in regulating ectopic endometrium growth.

Changes in the T-helper cytokine profile and in lymphocyte activation at the systemic and local levels in women with endometriosis.

OBJECTIVE: To estimate regulatory cytokine synthesis and lymphocyte activation in the peripheral blood and endometrial tissue of patients with endometriosis. DESIGN: Controlled clinical study. SETTING: Medical center. PATIENT(S): Fifteen women with laparoscopically diagnosed endometriosis; 20 gynecologically healthy women with previously documented fertility (control group). INTERVENTION(S): Peripheral venous blood sampling; laparoscopic collection of ectopic and matched eutopic endometrium. MAIN OUTCOME MEASURE(S): Messenger RNA (mRNA) for interleukin (IL)-2, IL-4, and IL-10 expression in peripheral and endometrium lymphocytes was assessed by real-time reverse transcriptase polymerase chain reaction; intracellular synthesis of these cytokines and lymphocyte phenotype profile were established by flow cytometry. RESULT(S): Both mRNA expression and intracellular synthesis of IL-4 and IL-10 were sharply increased in peripheral lymphocytes. The same changes were observed for IL-4 in ectopic endometrium of women with endometriosis. Simultaneously, elevation of the amount of pan-B cells, CD20+CD5+ B-1 cells, and activated HLA-DR+CD20+ B lymphocytes was observed in endometriosis lesions. Only an enhanced amount of B lymphocytes was seen in eutopic endometrium. CONCLUSION(S): Endometriosis development is accompanied by the activation of a T-helper type 2 immune response at the systemic and local levels. Our results support the hypothesis regarding the autoimmune nature of endometriosis and can explain the high level of autoantibody production in patients with endometriosis.

Local Epidermal Growth Factor Production in Women with Endometriosis.

The aim of the study was to elucidate the role of epidermal growth factor (EGF) in pathogenesis of endometriosis at different stages of disease. The level of EGF in peritoneal fluid (PF) of fertile and infertile women with endometriosis and in pooled supernatants of 24-h cultures of peritoneal macrophages was assessed by ELISA method. It was found that in fertile women with early stages of disease EGF level in PF did not differ from that in control group, but at the advanced stages of endometriosis the significant elevation of EGF concentration in PF was observed. The spontaneous production of EGF by peritoneal macrophages in this group was diminished. It might be suggested that elevation of EGF concentration in PF in this group is the result of secretory activity of endometriosis lesions and macrophages didn't regulate the local EGF production in fertile women. EGF level in PF of infertile women was increased only in women with minimal manifestations of endometriosis but not in women with advanced stages of the disease. Simultaneously the increase of EGF production by peritoneal macrophages of infertile women with advanced stages of endometriosis was shown. These facts give an evidence in favor of high level of macrophage activation and their participation in regulation of EGF level in peritoneal cavity in infertile women with endometriosis.

A Study of Peritoneal Immunocompetent Cells in External Genital Endometriosis.

The aim of the present investigation was to study the peculiarities of peritoneal lymphoid cell activation at the local level during external endometriosis and the possible role of secretory products of peritoneal macrophages in this process. Immunocompetent cells of peritoneal fluid from 16 women with endometriosis and 14 healthy women were studied by two-color flow cytometry method. It was found that endometriosis was associated with high level of expression of CD25 and HLA DR molecules and diminishment of Fas expression by CD3(+) subset of peritoneal lymphocytes. Experimentally it was established that supernatant of 24-h culture of peritoneal macrophages from women with endometriosis induced the expression of CD25 marker by CD4(+) but not CD8(+) cells and decreased Fas expression by both cell subsets in donor lymphocytes. It can be supposed that altered secretory function of peritoneal macrophages in situ impair the apoptosis of activated clones of T lymphocytes. Observed effect of macrophage secretory products was not associated with TNF-alpha production by peritoneal macrophages in the process of their cultivation.

Regulation of Expression of Lymphocyte Activation Markers by Neutrophil Factors Secreted during Pregnancy.

The work was outlined to elucidate contribution of soluble factors produced by peripheral neutrophils from healthy pregnant women and those with EPH-gestosis to regulation of expression of surface activation markers by donor lymphocytes. It was found out that in healthy pregnant women neutrophil factors stimulated expression of HLA-DR and CD25 antigens by T lymphocytes. In women with EPH-gestosis neutrophil factors, which comprised soluble antigen of leukocytes 2 (SAL-2), enhanced expression of CD25 antigen in B subset. Presumably, the effects were caused by activation of SAL-2 neutrophil secretion during EPH-gestosis. Impaired neutrophil secretion in women with EPH-gestosis may lead to activation of B lymphocytes through Th2 recruitment and triggering of the autoimmune reactions.