Endometriosis and human infertility: a new investigation into the role of eutopic endometrium.

BACKGROUND: Endometriosis is related to infertility even in the absence of mechanical alterations of the reproductive tract. Even though the pathogenesis of this phenomenon is still unclear, an impaired endometrial receptivity has been recently suggested. The aim of the present study was to investigate if endometriotic peritoneal fluids (EPF) could interfere with endometrial stromal cell (ESC) decidualization and if tumor necrosis factor (TNF)-alpha could be involved in the EPF effect. METHODS: Eutopic ESC were isolated from patients with or without endometriosis. ESC were treated with 17beta-estradiol 10(-8) M and 6alpha-methyl-17alpha-hydroxyprogesteroneacetate 2x10(-7) M for 16 days. In vitro decidualization was morphologically and biochemically assessed. We analysed whether ESC decidualization could be affected by EPF or peritoneal fluids from control patients (CPF), with or without soluble TNF-alpha receptor 1 (sTNFR-1). RESULTS: Compared with ESC from control patients, eutopic ESC from patients with endometriosis showed an impaired decidualization. Decidualization of normal ESC was morphologically normal but biochemically abnormal in the presence of EPF, which was able to decrease the secretion of decidualization markers. sTNFR-1 was able to partially counteract this effect. CONCLUSIONS: In endometriosis, the milieu surrounding the uterine cavity may be involved in impaired eutopic ESC decidualization, partially due to increased peritoneal levels of TNF-alpha.

Paracrine regulation of endometriotic tissue.

Endometriosis is a chronic estrogen-dependent gynecological disease, characterized by pelvic pain and infertility, defined as the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites. In the peritoneal cavity endometrial cells adhere, proliferate and induce an inflammatory response. Despite a long history of clinical and experimental research, the pathogenesis of endometriosis is still controversial. Abnormal immunological activation, the endocrine milieu and the peritoneal environment all dramatically affect endometriotic tissue function. Recent studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages and other immune cells that secrete various local products, such as growth factors and cytokines, which exert a paracrine action on endometriotic cells. Since the peculiar biological characteristics of eutopic endometrium from women with endometriosis differ from endometrium of normal subjects, an important role in the pathogenesis of this complex disease has been suggested. All of these factors contribute to enhanced proliferative and angiogenic activity and a number of functional and structural changes, resulting in the particular behavior of this tissue.

Ghrelin affects the release of luteolytic and luteotropic factors in human luteal cells.

CONTEXT: Ghrelin, well-known modulator of food intake and energy balance, is a rather ubiquitous peptide involved in several endocrine and nonendocrine actions. A possible as-yet-unknown role for ghrelin in modulating luteal function has been suggested because both ghrelin and its receptor (GRLN-R) have been immunohistochemically detected in human corpus luteum. OBJECTIVE: We first investigated GRLN-R mRNA expression in midluteal phase human luteal cells. Ghrelin effect on basal and human chorionic gonadotropin (hCG)-stimulated progesterone (P) release was then analyzed. Finally, we investigated whether ghrelin could affect luteal release of vascular endothelial growth factor (VEGF), prostaglandin (PG) E(2), both luteotropic factors, and PGF(2alpha), luteolytic modulator. Ghrelin effect on both basal and hypoxia-stimulated VEGF luteal expression was analyzed. METHODS: Human luteal cells were incubated for 24 h with ghrelin (10(-13) to 10(-7) m) or hCG (100 ng/ml) or CoCl(2) (10 microm), chemical hypoxia, or with hCG or CoCl(2) in combination with ghrelin. Both GRLN-R mRNA and VEGF mRNA were evaluated by real-time RT-PCR. PGs and P release was assayed by RIA, whereas VEGF release by ELISA. RESULTS: GRLN-R mRNA expression was demonstrated in human luteal cells. Both basal and hCG-stimulated P release was significantly decreased by ghrelin, which was able to reduce PGE(2) and increase PGF(2alpha) luteal release. Both basal and hypoxia-stimulated VEGF release was significantly decreased by ghrelin, which did not affect VEGF mRNA luteal expression. CONCLUSIONS: The present in vitro study provides the first evidence of a direct inhibitory influence of ghrelin on human luteal function.

Ghrelin in vitro modulates vasoactive factors in human umbilical vein endothelial cells.

OBJECTIVE: To determine whether Ghrelin could affect prostaglandins (PGs) and nitric oxide synthesis in human umbilical vein endothelial cells (HUVEC). The effect of Ghrelin on endothelial cell proliferation was also evaluated. DESIGN: In vitro research report. SETTING: Third-level referral academic centers, including molecular and cellular biology laboratories. PATIENT(S): Human umbilical cords were obtained from healthy female volunteers at term of uncomplicated pregnancies. INTERVENTION(S): HUVEC were cultured with Ghrelin (from 10(-11) to 10(-7) M). After 24 hours supernatants were collected and HUVEC were treated for total RNA extraction. MAIN OUTCOME MEASURE(S): In the culture medium PGs release was evaluated by RIA. Prostaglandin-endoperoxide synthase 2 (COX2) and both the constitutive and the inducible isoforms of nitric oxide synthases (ECNOS and INOS) mRNA expressions were evaluated by retrotranscriptase polymerase chain reaction. Endothelial cell proliferation was evaluated by bromo-deoxy-uridine incorporation and by cell counting. RESULT(S): Ghrelin negatively affected PGs release as well as COX2, ECNOS, and INOS mRNA expressions in HUVEC. Furthermore, Ghrelin increased bromo-deoxy-uridine incorporation in HUVEC without affecting cell counting. CONCLUSION(S): Our in vitro results allowed to hypothesize that Ghrelin could be involved in the modulation of vascular tone by affecting nitric oxide-related protein synthesis and PGs production in endothelial cells.

Regulation of vascular endothelial growth factor synthesis and release by human luteal cells in vitro.

CONTEXT: Vascular endothelial growth factor (VEGF) is essential for normal luteal development and function, but little is still known about the regulation of its production by human midluteal phase luteal cells. OBJECTIVE: We investigated whether human chorionic gonadotropin (hCG) or local factors, including chemical hypoxia, IGF-I and IGF-II, prostaglandin (PG)E(2), and PGF(2alpha) prevail in modulating VEGF mRNA and protein production in human midluteal phase luteal cells. The effect of progesterone (P) on luteal VEGF mRNA expression and protein secretion was also evaluated. Finally, we investigated whether VEGF could directly affect luteal P secretion. INTERVENTIONS: In human midluteal phase luteal cells, VEGF mRNA expression was evaluated by semiquantitative RT-PCR, whereas VEGF and P release was evaluated by ELISA and RIA, respectively. RESULTS: hCG was unable to significantly affect luteal VEGF mRNA and protein synthesis, which in turn was significantly increased by both chemical hypoxia and IGFs. Conversely, VEGF mRNA and protein production was reduced by PGs and P. Finally, VEGF did not affect P luteal secretion. CONCLUSIONS: Our results suggest that local ovarian factors, rather than hCG, predominate in regulating VEGF mRNA and protein production by human midluteal phase luteal cells. For VEGF, a lack of a direct luteal steroidogenic effect was also demonstrated.

Effects of nicotine on human luteal cells in vitro: a possible role on reproductive outcome for smoking women.

We investigated the effect of nicotine and its methylated metabolite, N-methyl-nicotine (M-nicotine), on human luteal cells by measuring release of progesterone and prostaglandins (PGs) from cultured cells and by testing gene expression of vascular endothelial growth factor (VEGF), an angiogenic factor strictly involved in luteal pathophysiology. Primary cultures of human luteal cells were treated for 24 h with nicotine and M-nicotine (from 10(-6) to 10(-11) M) either alone or combined with hCG (25 ng/ml); progesterone and PGs were assayed in the culture medium. In another group of experiments, luteal cells were treated for 24 h with nicotine and M-nicotine (10(-7) M) to perform reverse transcriptase-polymerase chain reaction on VEGF mRNA. Nicotine and M-nicotine negatively affected basal luteal steroidogenesis at all tested concentrations, but neither was able to affect hCG-induced progesterone release. Both substances were able to significantly increase PGF2alpha release from luteal cells, with a dose-related efficacy for M-nicotine. On the contrary, PGE2 release was significantly inhibited by both nicotine and its metabolite. Finally, nicotine was able to increase VEGF mRNA expression significantly, whereas M-nicotine was not. In conclusion, nicotine and M-nicotine can induce a sort of luteal insufficiency by inhibiting progesterone release, probably through modulation of the PG system.

Effects of insulin-like growth factor I and II on prostaglandin synthesis and plasminogen activator activity in human umbilical vein endothelial cells.

IGFs seem to contribute to the endothelial dysfunction observed in some vascular diseases. Because locally increased IGFs levels were detected in the preeclamptic fetoplacental unit, we hypothesized their involvement in the dysregulation of fibrinolysis and vascular tone typically observed in the fetoplacental compartment in this pregnancy disease. Therefore, in human umbilical vein endothelial cells (HUVECs), the potential effect of IGFs on the synthesis of plasminogen activators (PAs), PA inibitor-1 (PAI-1), and vasodilator and vasoconstrictor prostaglandins (PGs) was investigated. Moreover, in HUVECs treated with IGFs, the expression of cyclooxygenase (COX)-2, the rate-limiting enzyme in PG synthesis, was evaluated.HUVECs were treated for 24 h with IGFs (1-100 ng/ml) or IL-1beta (0.1 ng/ml). PA, PAI-1, and COX-2 mRNA was determined by RT-PCR and PG release and PA activity by RIA and colorimetric assay, respectively.We demonstrated an inhibition of urokinase-type PA activity and a 50% reduction of urokinase-type PA mRNA in HUVECs treated with IGFs. No effect was seen on PAI-1. Finally, both IGFs significantly decreased all PGs tested and COX-2 mRNA, whereas, as expected, IL-1beta had an opposite effect. In conclusion, our results suggest for IGFs a potential involvement in the endothelial dysfunction observed in preeclamptic fetoplacental unit.

Endometrial evaluation in superovulation programs: relationship with successful outcome.

It is well known that an adequate endometrial receptivity is required for successful implantation in both natural and assisted reproductive cycles. In particular, a brief "implantation window", during which endometrium undergoes anatomical and molecular changes necessary for embryo implantation, has been observed. The hormonal treatment applied to induce ovulation seems to be able to modify the normal development of the prenidatory endometrium, with possible negative effect on the implantation rate. For this reason, several attempts have been made to identify specific markers of endometrial receptivity, useful for predicting implantation outcome in clinical practice. Even if different histological, immunohistochemical, and ultrasonographic parameters are studied, none unfortunately has been univocally shown to be predictive of pregnancy outcome. Therefore, the evaluation of endometrial receptivity remains a challenge in clinical practice.

In vitro regulation of beta1 and beta3 integrin subunits in endometrial epithelial cells from normal endometrium.

OBJECTIVE: To evaluate the effect of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) on integrin expression. DESIGN Cultures of endometrial epithelial cells from normal endometrium. SETTING: All endometrial specimens were obtained from the Obstetrics and gynecology Department of the Catholic University, Rome, Italy. PATIENTS: Four patients were normal menstrual cycles undergoing operative laparoscopy for non-endometrial problems. INTERVENTION: Endometrial samples were collected by uterine courettings. MAIN OUTCOME MEASURE: Immunocytochemistry for beta1 and beta3 integrin subunits. RESULTS: PGE2 clearly enhances both beta1 and beta3 integrin subunit expression. IL-1beta seem to slightly increase only beta3 subunit expression. CONCLUSIONS: In light of the critical role played by eicosanoids in endometrial differentiation, we suggest that PGE2 is also involved in local paracrine regulation of integrin expression.

The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells.

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.

Interleukin-1 beta stimulates progesterone production by in vitro human luteal cells: evidence of a mediatory role of prostaglandins.

We have investigated whether IL-1 beta, a cytokine with an important role in ovarian physiology, is also involved in progesterone (P) synthesis in human luteal cells, and whether this effect is mediated via the cyclooxygenase (COX) pathway. Human luteal cells were cultured for 24 h in the presence of IL-1 beta (0.01-10 ng/ml), given alone or in combination with human chorionic gonadotropin (100 ng/ml), indomethacin (1 micro g/ml), or P (100 ng/ml). We observed a significant increase in prostaglandin (PG)release after IL-1 beta treatment; the cytokine was more effective on PGE(2) than PGF(2 alpha) release. The effect of IL-1 beta was abolished by human chorionic gonadotropin, which had no action on basal PG levels when given alone; in contrast, P reduced basal, but not IL-1 beta-stimulated, PG production. Treatment with the human IL-1 receptor antagonist was associated with a decrease in both basal and IL-1 beta-stimulated PG production. Moreover, IL-1 beta induced a concentration-dependent increase in P production and release, an effect counteracted by the COX inhibitor indomethacin. In conclusion, our data show the ability of IL-1 beta to influence P secretion via the COX pathway, thereby suggesting a possible luteotropic role in human ovary based on an autocrine-paracrine mechanism.