The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions

Studies were performed to elucidate the possible relationship between microvessel density, proliferative activity and angiogenesis in eutopic endometrium from women with and without endometriosis and peritoneal endometriotic lesions. The question whether changes in these parameters in endometriotic lesions were reflected by the level of vascular endothelial growth factor-A (VEGF-A) in serum and peritoneal fluid was also studied. Biopsy specimens of both eutopic endometrium and peritoneal endometriotic lesions from women with endometriosis (n = 25) as well as eutopic endometrium from women without endometriosis (n = 14) were analysed immunohistochemically regarding microvessel density, proliferative activity, and expression of VEGF-A and its receptors vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2) in stroma, glands and blood vessels. The VEGF-A concentration was measured in peritoneal fluid and serum. Secretory phase eutopic endometrium from women with endometriosis had significantly higher microvessel density, expression of VEGF-A in glandular epithelium and VEGFR-2 in endometrial blood vessels than those from women without endometriosis. Endometriotic lesions with high proliferative activity had a higher microvessel density and showed higher vascular expression of VEGFR-2 as well as being accompanied by higher levels of VEGF-A in peritoneal fluid and serum, compared with lesions with low proliferative activity. In conclusion, there seems to be a dysregulation of angiogenic activity in the eutopic endometrium of women with endometriosis and endometriotic lesions with high proliferative activity were accompanied by higher local angiogenic activity and higher levels of VEGF in serum and peritoneal fluid.     

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus)

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.     

Intravascular neutrophils partially mediate the endometrial endothelial cell proliferative response to oestrogen in ovariectomised mice

The aim of this study was to investigate the role of intravascular neutrophils in initiating endothelial cell proliferation following oestrogen treatment in ovariectomised mouse endometrium. Uterine tissues were collected from ovariectomised C57/CBA female mice 24 h after oestrogen treatment with or without systemic neutrophil depletion. Neutropenia was achieved with either an in-house anti-neutrophil serum (ANS) or Gr-1 monoclonal antibody. All mice received an i.p. injection of bromodeoxyuridine (BrdU) 4 h prior to dissection to allow visualisation of proliferating cells using immunocytochemistry. Endometrial sections were immunostained for BrdU, vascular endothelial growth factor (VEGF), and neutrophils (using ANS). Oestrogen treatment of ovariectomised mice significantly increased the number of intravascular neutrophils, whereas induction of neutropenia with either ANS or Gr-1 in conjunction with oestrogen treatment prevented this increase. Oestrogen treatment of ovariectomised mice also significantly increased the number of intravascular VEGF-positive cells; however, whereas induction of neutropenia with ANS significantly reduced this increase, Gr-1 did not. In both studies, neutropenia significantly reduced, but did not eliminate, the amount of endometrial endothelial cell proliferation. These results suggest a role for neutrophils in endometrial angiogenesis following acute oestrogen treatment; however, the presence of VEGF-positive cells even after induction of neutropenia suggests that more than one type of leukocyte may be involved.     

Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy

Immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (flt-1) and kinase insert domain-containing region (KDR), was performed on human endometrium obtained from patients with normal menstrual cycles, patients given oestrogen and progesterone, and women in early pregnancy. Intense immunostaining of VEGF was observed in both glandular epithelial and stromal cells during the mid-secretory phase; the immunostaining intensity was increased by administration of oestrogen plus progesterone and strong immunostaining was observed in decidual cells of early pregnancy. In addition to the immunostaining in vascular endothelial cells, strong KDR immunostaining was observed in glandular epithelial cells and in decidualized stromal cells induced by administration of oestrogen plus progesterone, whereas flt-1 immunostaining was negligible. Strong immunostaining for flt-1 and KDR was found in both vascular endothelial cells and decidual cells in early pregnancy. Endometrial stromal cells isolated from proliferative phase endometrium were incubated with oestrogen (10(-8) mol l-1) and medroxyprogesterone acetate (MPA; 10(-6) mol l-1) for 18 days to study the regulation of VEGF, flt-1 and KDR in endometrial stromal cells by oestrogen and progesterone. Expression of VEGF and KDR mRNAs was increased significantly by oestrogen and MPA, accompanied by decidualization, whereas flt-1 mRNA expression was not affected. In conclusion, VEGF and its receptors may play important roles in implantation and maintenance of pregnancy.     

Regulation of homeobox A10 expression in the primate endometrium by progesterone and embryonic stimuli

Homeobox A10 (HOXA10), a member of abdominal B subclass of homeobox genes, is responsible for uterine homeosis during development. Intriguingly, in the adult murine uterus, HOXA10 has been demonstrated to play important roles in receptivity, embryo implantation, and decidualization. However, the roles of HOXA10 in the primate endometrium are not known. To gain insights into the roles of HOXA10 in the primate endometrium, its expression was studied in the endometria of bonnet monkey (Macaca radiata) in the receptive phase and also in the endometria of monkeys treated with antiprogestin onapristone (ZK98.299) or in conception cycle where the presence of preimplantation stage blastocyst was verified. In addition, the mRNA expression of HOXA11 and insulin-like growth factor-binding protein 1 (IGFBP1) was evaluated by real-time PCR in these animals.The results revealed that HOXA10 in the luteal phase primate endometrium is differentially expressed in the functionalis and the basalis zones, which is modulated in vivo by progesterone and also by the signals from the incoming embryo suggesting the involvement of HOXA10 in the process of establishment of pregnancy in primates. In addition, the results also demonstrated that the expression of IGFBP1 but not HOXA11 is coregulated with HOXA10 in the endometria of these animals. The pattern of changes in the expression of HOXA10 in response to the two stimuli suggests that endometrial receptivity and implantation not only requires a synchrony of maternal and embryonic signaling on endometrial cells in the primates but there also exists a controlled differential response among the cells of various uterine compartments.     

Bioactive Lipids in Pathological Retinopathy

Diabetic retinopathy is a common condition that occurs in patients with diabetes with long-standing hyperglycemia that is characterized by inappropriate angiogenesis. This pathological angiogenesis could be a sort of physiological proliferative response to injury by the endothelium. Recent studies suggested that reactive oxygen species (ROS) play a significant role in this angiogenesis. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that plays a significant role in diabetic retinopathy. The interaction between VEGF and ROS, and theirs in turn with pro- and anti-inflammatory cytokines and anti-inflammatory bioactive lipid molecules such as lipoxins, resolvins, protectins, and maresins is particularly relevant to understand the pathophysiology of diabetic retinopathy and develop future therapeutic interventions.     

The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice

The role of progesterone (and oestrogen) in endometrial angiogenesis remains controversial. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell proliferation in ovariectomized mice. Uteri were collected on days 1 to 4 of pregnancy when circulating progesterone concentrations were increasing, prior to implantation. Before dissection, mice were injected with bromodeoxyuridine (BrdU) enabling proliferating endothelial cells to be quantified with CD31/BrdU double-immunohistochemistry. There was a significant increase in proliferating endothelial cells on day 3 of pregnancy when plasma progesterone also increased. To determine if this endothelial cell proliferation was due to progesterone, an experiment was performed on ovariectomised mice. One group was treated with a single oestradiol injection on day 8 after ovariectomy, followed by a no-treatment day and three consecutive daily injections of progesterone. Other groups were treated with either the vehicle, oestradiol or progesterone injections only; all were dissected on day 13 following ovariectomy. Unexpectedly, mice treated with progesterone-only had the highest amount of endothelial cell proliferation and oestrogen priming was found to significantly reduce this progesterone-induced endothelial cell proliferation. To determine if this proliferation is mediated by vascular endothelial growth factor (VEGF), a further experiment in which VEGF anti-serum was administered concurrently with the progesterone injections was performed. Endothelial cell proliferation was reduced but not abolished suggesting progesterone-induced endometrial angiogenesis is only partly mediated by VEGF. Results indicate that oestrogen priming is not required for progesterone to stimulate endometrial endothelial cell proliferation and that oestrogen inhibits progesterone-induced angiogenesis in ovariectomised mice.     

Expression of CD105 (endoglin) in arteriolar endothelial cells of human endometrium throughout the menstrual cycle

The cellular mechanisms underlying normal and pathological endometrial bleeding are not well understood, although abnormalities in the structure of endometrial blood vessels may lead to menstrual disorders. Endothelial cells in different organs are heterogeneous and differ in structure, function, antigen composition, metabolic properties and responses to growth factors. Immunostaining was performed with anti-CD105, CD31, CD34 and von Willebrand factor (vWF), and lectin binding with Ulex europaeus agglutinin 1 (UEA 1), Bandeieraea simplicifolia agglutinin 1 (BS 1), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) to characterize endothelial cells in human endometrium throughout the menstrual cycle. Serial sections fixed with formalin were stained with primary antibodies and lectins after antigen retrieval. Positive staining for CD31, CD105 and vWF was confined to the vascular endothelium. Endothelial expression of CD31 was observed in all types of vessel, including single cells, and strong staining was found during the early proliferative and mid-secretory phases. Anti-vWF stained arterioles and veins, but there was little positive staining of capillaries. In contrast, staining for CD105 was confined to the arterioles. Although anti-CD34 strongly stained endothelial cells of small vessels and capillaries, staining was also observed on some non-endothelial stromal cells. Strong positive staining for UEA 1 was observed in endothelial cells of all types of vessel throughout the menstrual cycle. Binding of PNA, DBA and BS 1 was confined to the apical region of glandular epithelial cells. This study demonstrates that the differential binding of anti-CD31, CD34, CD105, vWF and UEA 1 distinguishes between endometrial populations of endothelial cells.     

Oestrogen and progesterone receptors in the marmoset endometrium: changes during the ovulatory cycle, early pregnancy and after inhibition of vascular endothelial growth factor, GnRH or ovariectomy

Marmosets are widely used, but detailed studies on localisation of endometrial oestrogen receptors alpha and beta (ER alpha and ER beta ), and the progesterone receptor (PR) are lacking. These receptors were localised and semi-quantitatively analysed throughout the ovulatory cycle, weeks 2, 3 and 4 of pregnancy and after treatment with GnRH antagonist, vascular endothelial growth factor (VEGF) Trap or ovariectomy. The PR in epithelial cells increased markedly between the mid- and late proliferative phases before declining in the mid-secretory phase and pregnancy. PR in stromal cells was present throughout the cycle and levels were maintained in pregnancy. ER alpha was present at the mid-proliferative phase and increased in glands at the late proliferative and early secretory phases, before declining at the late secretory phase and week 4 of pregnancy. Stromal ER alpha showed a similar trend, but decreased earlier, by the mid-secretory phase. ER beta was highly expressed in epithelial cells throughout the cycle and in pregnancy. In stroma, increases in ER beta expression were observed at the late proliferative phase with the staining index decreasing by half as the secretory phase progressed and in pregnancy. GnRH antagonist, VEGF Trap or ovariectomy caused significant reductions in PR and ER beta expression, but not in ER alpha when compared with the late proliferative phase of the normal cycle. Endothelial cells expressed ER beta , but not ER alpha or PR. It is concluded that the steroid receptor profile in the marmoset endometrium is generally similar to the human and should provide a useful model for studies on hormonal manipulation of the endometrium.     

A novel physiological culture system that mimics luteal angiogenesis

Luteal inadequacy is a major cause of poor embryo development and infertility. Angiogenesis, the formation of new blood vessels, is an essential process underpinning corpus luteum (CL) development and progesterone production. Thus, understanding the factors that regulate angiogenesis during this critical time is essential for the development of novel strategies to alleviate luteal inadequacy and infertility. This study demonstrates the development of a physiologically relevant primary culture system that mimics luteal angiogenesis. This system incorporates all luteal cell types (e.g. endothelial, steroidogenic cells, fibroblasts and pericytes). Using this approach, endothelial cells, identified by the specific marker von Willebrand factor (VWF), start to form clusters on day 2, which then proliferate and develop thread-like structures. After 9 days in culture, these tubule-like structures lengthen, thicken and form highly organized intricate networks resembling a capillary bed. Development of the vasculature was promoted by coating wells with fibronectin, as determined by image analysis (P<0.001). Progesterone production increased with time and was stimulated by LH re-enforcing the physiological relevance of the model in mimicking in vivo luteal function. LH also increased the area stained positively for VWF by twofold (P<0.05). Development of this endothelial cell network was stimulated by fibroblast growth factor 2 and vascular endothelial growth factor A, which increased total area of VWF positive staining on day 9, both independently (three- to fourfold; P<0.01) and in combination (tenfold; P<0.001). In conclusion, the successful development of endothelial cell networks in vitro provides a new opportunity to elucidate the physiological control of the angiogenic process in the developing CL.     

Ovarian VEGF(165)b expression regulates follicular development, corpus luteum function and fertility

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.     

The effects of ergot and non-ergot-derived dopamine agonists in an experimental mouse model of endometriosis

Implantation of a retrogradely shed endometrium during menstruation requires an adequate blood supply, which allows the growth of endometriotic lesions. This suggests that the development of endometriosis can be impaired by inhibiting angiogenesis. The growth of endometriotic foci is impaired by commercial oncological antiangiogenic drugs used to block vascular endothelial growth factor (VEGF) signaling. The dopamine agonist cabergoline (Cb2) inhibits the growth of established endometriosis lesions by exerting antiangiogenic effects through VEGFR2 inactivation. However, the use of ergot-derived Cb2 is associated with an increased incidence of cardiac valve regurgitation. To evaluate the potential usage of non-ergot-derived dopamine agonists for the treatment of human endometriosis, we compared the efficacy of quinagolide with that of Cb2 in preventing angiogenesis and vascularization in a heterologous mouse model of endometriosis. Nude mice whose peritoneum had been implanted with eutopic human endometrial fragments were treated with vehicle, 50 μg/kg per day oral Cb2, or 50 or 200 μg/kg per day quinagolide during a 14-day period. At the end of the treatment period, the implants were excised in order to assess lesion size, cell proliferation, degree of vascularization, and angiogenic gene expression. Neoangiogenesis was inhibited and the size of active endometriotic lesions, cellular proliferation index, and angiogenic gene expression were significantly reduced by both dopamine agonists when compared with the placebo. Given that Cb2 and quinagolide were equally effective in inhibiting angiogenesis and reducing lesion size, these experiments provide the rationale for pilot studies to explore the use of non-ergot-derived dopamine agonists for the treatment of endometriosis in humans.     

Regulation of endometrial endothelial cell proliferation by oestrogen and progesterone in the ovariectomized mouse

Although the endometrial epithelial and stromal cell response to oestrogen and progesterone is well characterized, relatively little is known about the endothelial cell response. The aim of this study was to investigate the time course of endometrial endothelial cell proliferation in response to a specific regimen of oestrogen and progesterone, and to compare it with the stromal and epithelial cell response in mouse endometrium. Adult female mice were ovariectomized to induce endometrial regression. After 7 days, hormonal treatments were given according to the following regimen: days 1-3: 100 ng oestradiol; days 4-6: 10 ng oestradiol and 500 microg progesterone; and day 7: 100 ng oestradiol and 500 microg progesterone. On each day of hormonal treatment, mice (n = 5) were injected with bromodeoxyuridine and perfusion fixed 4 h later with buffered formalin. Proliferating endometrial cells were detected by monoclonal antibody against bromodeoxyuridine, and endothelial cells were detected by antibody to CD31. At day 7 after ovariectomy few proliferating cells were found in the endometrium. After 1 day of oestrogen treatment, significant proliferation was detected in the endothelial cells (0.0% versus 16.1 +/- 1.2%, P < 0.001). In contrast to the rapid response of the vasculature, glandular epithelial proliferation increased only after 2 days of oestrogen treatment (7.6 +/-1.3% versus 18.8 +/- 2.4%, P < 0.05). Progesterone with low dose oestrogen treatment tended to reduce epithelial and endothelial cell proliferation compared with the effect of high dose oestradiol alone. A combination of progesterone with high dose oestrogen induced higher rates of endothelial cell proliferation than did any other treatment (20.8 +/- 3.2%). These results demonstrate that oestrogen induces rapid proliferation of endometrial endothelial cells, indicating that vascular growth apparently precedes endometrial tissue remodelling. These data also demonstrate that the proliferative response of endometrial endothelial cells to oestrogen and progesterone is different from that of either epithelial or stromal cells.     

Capillary angiogenesis and degeneration in bovine ovarian antral follicles

Angiogenesis and capillary degeneration are both evident during ovarian follicle growth. However, the characteristics and distribution of thecal capillary proliferative and degenerative structures have not been fully defined. Indeed, the role of thecal microvasculature changes in follicular atresia is still a matter of debate. The present study examined the distribution of thecal capillary changes occurring during follicular growth and related the changes to capillary morphology (by scanning electron microscopy, SEM, on bovine ovarian corrosion casts) with the incidence of capillary apoptosis (TdT-mediated dUTP nick end-labelling, TUNEL) and follicular status (as confirmed by follicular fluid steroid concentrations). SEM demonstrated well-perfused vascular plexuses of small to large antral follicles with structural and functional changes to capillaries. Angiogenesis was evident mainly in the apical part of the inner capillary layer of medium follicles and the middle or basal part of the inner capillary layer of dominant follicles that exhibited high oestradiol:progesterone ratios. Degenerative capillaries were observed mainly in the outer vascular layers of small follicles, and in the inner and outer vascular layers of medium antral follicles. Although apoptotic structures were present only in the outer capillaries of the theca interna of morphologically healthy antral follicles, atretic follicles showed apoptotic structures in both the outer and inner thecal capillary layers. These results show that angiogenesis increases during bovine follicular growth and occurs unevenly in different inner theca regions of the follicles. The differential angiogenic and degenerative response of theca interna capillaries may reflect differences in the microenvironment of the follicles, which in turn determine the fate of the follicles (continued growth versus atresia).     

Angiogenesis and microvascular development in the marmoset (Callithrix jacchus) endometrium during early pregnancy

The aim of the study was to describe and quantify the changes in the maternal vasculature and angiogenesis during early pregnancy in the marmoset endometrium using bromodeoxyuridine (BrdU) to identify proliferating cells, CD31 to label endothelial cells and dual staining to identify proliferating endothelial cells. Non-pregnant animals from mid- and late secretory stages were studied and compared with pregnant animals at weeks 2, 3 and 4 of pregnancy. Qualitative and morphometric analyses of angiogenesis and vascular area were performed. The results show that pregnancy is associated with increasing angiogenesis in the upper zone of the endometrium, becoming significantly increased at 3 weeks. This is associated with an increase in the vessel area and diameter in this zone. These results provide the platform from which to design studies in which specific angiogenic factors can be targeted in vivo during early pregnancy in order to determine their role in regulating these vascular changes.     

Mast cell degranulation in rat uterine cervix during pregnancy correlates with expression of vascular endothelial growth factor mRNA and angiogenesis

Vascular growth of the uterine cervix during pregnancy is associated with mast cell (MC) degranulation. To better understand the mechanism underlying this process, uterine cervices of intact pregnant rats were dissected and endothelial cell proliferation was measured by a bromodeoxyuridine incorporation technique. Total vascular endothelial growth factor (VEGF) mRNA expression and the relative abundance of VEGF splice variants (120, 164, and 188) were determined by RT-PCR. VEGF protein expression was evaluated by immunohistochemistry. To investigate the role of MCs on cervical angiogenesis, a second set of pregnant animals were treated with an MC stabilizer (disodium cromoglycate) to inhibit MC degranulation. Furthermore, 17beta-estradiol (E(2)) serum levels were established by RIA. In intact pregnant rats, VEGF mRNA expression was positively correlated with endothelial cell proliferation and circulating E(2) levels. All selected splice variants of VEGF gene were detected and their relative abundance did not show any change throughout pregnancy. Animals treated with disodium cromoglycate showed a decrease in endothelial cell proliferation and in VEGF mRNA expression compared with controls. Relative abundance of VEGF mRNA splice variants and E(2) serum levels showed no differences between these experimental groups. These results show a time-dependent correlation between VEGF mRNA expression and E(2) serum levels in the uterine cervix of intact pregnant rats, while MC stabilizer-treated animals reduced the VEGF expression without modifying E(2) serum levels. We suggest that cervical angiogenesis during pregnancy could be regulated by a mechanism which involves endogenous E(2) and chemical mediators stored in MC granules via a VEGF-dependent pathway.     

Temporal expression and signalling of prostacyclin receptor in the human endometrium across the menstrual cycle

Prostacyclin (PGI(2)) synthesis and function in the human uterus has been implicated in the regulation of the process of normal and dysfunctional menstruation. PGI(2) synthesis is elevated during normal menstruation and is also associated with blood loss in women who suffer from heavy menses. This study was designed to outline further the role of PGI(2) in menstruation by investigating the temporal pattern and site of expression of prostaglandin I synthase (PGIS) and the prostacyclin receptor (IP receptor) in the non-pregnant human endometrium across the menstrual cycle. Quantitative RT-PCR demonstrated increased expression of PGIS and IP receptor during the menstrual phase of the cycle compared with all other phases (P < 0.05). Furthermore, PGIS and IP receptor were localised to the glandular epithelium, stromal and endothelial cells in the basal and functional layers of the endometrium. Functionality of the IP receptor in the human endometrium was assessed by measuring cAMP generation following treatment with 100 nmol l(-1) of the PGI(2) analogue, iloprost. cAMP generation was significantly higher in endometrial tissue collected during the proliferative compared with the secretory phase of the menstrual cycle (P < 0.05). In conclusion, this study has confirmed increased expression and signalling of PGIS and IP receptor during the menstrual phase and outlines a potential autocrine/paracrine role for PGI(2) on several cellular compartments in the endometrium including the endothelium. This may underscore a pivotal role for PGI(2) receptor signalling in normal and dysfunctional menstruation.