Expression of 11 beta-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies

Interconversion of active and inactive glucocorticoids, e.g. cortisol (F) and cortisone (E) is catalysed by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) which exists as two isoforms. We have used human placental bed biopsies and an in-vitro cytotrophoblast cell culture system to examine the expression and activity of the 11 beta-HSD isoforms along with that of the glucocorticoid and mineralocorticoid receptors (GR and MR). Immunohistochemistry localized 11 beta-HSD1 to decidualized stromal cells and 11 beta-HSD2 to villous cytotrophoblast, syncytiotrophoblasts and trophoblast cells invading the placental bed and maternal vasculature. In primary cultures of human cytotrophoblast, 11 beta-HSD2, GR and MR mRNA were expressed. Low levels of 11 beta-HSD1 mRNA were noted in these cultured cells, but could be explained on the basis of contaminating, vimentin-positive decidual stromal cells (< or =5%). Enzyme activity studies confirmed the presence of a high-affinity, NAD-dependent dehydrogenase activity (K(m) 137 nmol/l and V(max) 128 pmol E/h/mg protein), indicative of the 11 beta-HSD2 isoform. No reductase activity was observed. The presence of functional MR and GR was determined using Scatchard analyses of dexamethasone and aldosterone binding (MR K(d) 1.4 nmol/l B(max) 3.0; GR K(d) 6.6 nmol/l B(max) 16.2 fmol/ng protein). The expression of 11 beta-HSD1 in maternal decidua and 11 beta-HSD2 in adjacent trophoblast suggests an important role for glucocorticoids in determining trophoblast invasion. The presence of the MR within trophoblast indicates that some of the effects of cortisol could be MR- rather than GR-mediated.     

Regulatory role of angiotensin II on progesterone production by cultured human granulosa cells. Expression of angiotensin II type-2 receptor

The role of angiotensin II (AngII) in ovarian steroidogenesis is not clearly understood. In order to study its action on progesterone synthesis and to determine which receptor subtype is involve, granulosa cells obtained from women undergoing in-vitro fertilization were cultured for 2 or 4 days and then incubated in the presence of AngII (10(-7) M) with or without human chorionic gonadotrophin (HCG, 10 IU/ml) for 3 or 18 h. In cells cultured for 2 days, incubation with AngII decreased progesterone secretion by 36%, and inhibited activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) by 87% (P < 0.05), although its expression was not significantly reduced. However, in cells cultured for 4 days, progesterone production was enhanced by incubation with AngII (38%), and no change was observed in 3 beta-HSD expression. Both inhibitory and stimulatory effects were dose- dependent. Progesterone secretion was increased (93%) by incubation with HCG of cells cultured for 4, but not for 2 days, and no potentiation was observed with AngII. Treatment with PD123177 completely blocked the action of AngII and decreased the HCG-stimulated secretion of progesterone by 27%. Angiotensin type-2 (AT2) receptor mRNA was expressed in cells cultured for 4 days. In conclusion, AngII showed a regulatory role in in-vitro progesterone production by human granulosa luteinized cells, modulating the activity of 3 beta-HSD. It is likely that these actions may be mediated via membrane receptors, possibly of the AT2 receptor family.      

Potential regulation of inflammation in the lung by local metabolism of hydrocortisone

Human lung tissue converts hydrocortisone to cortisone by the action of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). Since cortisone is inactive as an antiinflammatory steroid, the action of this enzyme may regulate the local antiinflammatory effects of glucocorticoids in the lung. Minced human lung tissue (100 mg in 500 microliters medium) metabolized approximately 50% of added [3H]hydrocortisone within 2 h in most lung specimens. Metabolism was linear during this period and was found to occur over a broad range of concentrations of hydrocortisone (1 to 1,000 nM). Metabolism of hydrocortisone was not observed in minced pulmonary blood vessels or airways (2 to 3 mm), and pleura (containing some adherent parenchyma) had less activity than parenchyma. Cultured human tracheal epithelial cells metabolized hydrocortisone, while umbilical vein endothelial cells did not. Since glycyrrhizin, glycyrrhetinic acid, and carbenoxolone have antiinflammatory properties and have recently been shown to interfere with steroid metabolism in renal tissues, their effects on 11 beta-HSD in human lung tissue have been tested. Conversion of hydrocortisone to cortisone by lung tissue was inhibited by glycyrrhetinic acid (IC50, approximately 2.5 x 10(-8) M) and carbenoxolone (1.5 x 10(-7) M), but not glycyrrhizin (greater than 10(-5) M). It is proposed that inhibition of the metabolism of hydrocortisone by 11 beta-HSD may partially explain the known antiinflammatory actions of orally administered glycyrrhetinic acid and carbenoxolone and that intrapulmonary administration of these compounds may produce antiinflammatory effects targeted to the lung.     

11 beta-Hydroxysteroid dehydrogenase: a link between the dysregulation of cortisol metabolism and hypertension

Endocrine pathology is a well-recognised and important cause of human hypertension. Recent research has highlighted the role of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in the development of certain forms of hypertension. This enzyme, which exists as two genetically unique isoforms, 11 beta-HSD1 and 11 beta-HSD2, is responsible for the interconversion of biologically active cortisol with its inactive 11-oxo derivative, cortisone. Congenital deficiency of 11 beta-HDS2 results in inappropriate activation of the renal mineralocorticoid receptor by cortisol, leading to hypertension, hypokalaemia and metabolic alkalosis. Several authors have postulated a link between changes in 11 beta-HSD activity and the development of certain forms of essential hypertension. The existence of endogenous inhibitors of the enzyme provides compelling evidence in favour of this hypothesis, but few have been able to demonstrate a clear link between inhibition of 11 beta-HSD2 activity and hypertension by this mechanism. Similarly, several authors have suggested a relationship between reduced placental 11 beta-HSD2 activity, low birth weight with high placental weight, and the development of hypertension in adulthood. However, no clear evidence to suggest a direct correlation between birth weight, placental weight and 11 beta-HSD2 activity has been demonstrated. While the role of 11 beta-HSD in the development of hypertension remains controversial, an understanding of the interplay of this enzyme with both mineralocorticoid and glucocorticoid receptors undoubtedly will yield data that will clarify this complex field.     

Kit ligand and c-Kit have diverse roles during mammalian oogenesis and folliculogenesis

Paracrine signalling between the oocyte and its surrounding somatic cells is fundamental to the processes of oogenesis and folliculogenesis in mammals. The study of animal models has revealed that the interaction of granulosa cell-derived kit ligand (KL) with oocyte and theca cell-derived c-Kit is important for multiple aspects of oocyte and follicle development, including the establishment of primordial germ cells within the ovary, primordial follicle activation, oocyte survival and growth, granulosa cell proliferation, theca cell recruitment and the maintenance of meiotic arrest. Though little is known about the specific roles of KL and c-Kit during human oogenesis, the expression profiles for KL and c-Kit within the human ovary suggest that they are also functionally relevant to female fertility. This review details our current understanding of the roles of KL and c-Kit within the mammalian ovary, with a particular focus on the functional diversity of this receptor-ligand interaction at different stages of oocyte and follicle development.     

Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells

The human ovarian surface epithelium (hOSE) is a single layer of mesothelial-type primitive epithelial cells that are potential estrogen targets. It has been reported that hOSE cells can produce estrogen. However, the mechanisms that regulate estrogen level(s) in hOSE cells are not yet known. To elucidate the enzymes involved in these reactions, we examined gene expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in primary hOSE (POSE) and OSE2a cells using RT-PCR. We found that POSE cells and cells of the immortalized hOSE line, OSE2a, bidirectionally converted estrone (E1) and 17beta-estradiol (E2). Both cell types expressed mRNA for 17beta-HSD type 1 (17beta-HSD1), suggesting that the enzyme is involved in the E1 to E2 conversion. Interestingly, both cells expressed 17beta-HSD4 mRNA but not 17beta-HSD2 mRNA. We prepared an antibody against the carboxyl terminal of 17beta-HSD4 (anti-17beta-HSD4 antibody), which recognized the 80 and 48 kDa proteins in POSE and OSE2a cells based on immunoblot analysis. Furthermore, immunohistochemical study revealed the presence of 17beta-HSD4 in hOSE cells in the human ovary. These results suggest that 17beta-HSD4 is involved in estrogen inactivation and may protect against an excessive accumulation of E2 in hOSE cells.     

Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells

BACKGROUND: Human follicular fluid contains several substances, such as cytokines and growth factors, which may affect follicular growth and maturation. The present study examines the relative contribution of macrophages and granulosa cells in the production of vascular endothelial growth factor (VEGF) and adrenomedullin in the human ovulatory follicle. METHODS: Both follicular fluid samples and blood samples were obtained at the time of oocyte retrieval following ovarian stimulation from 20 women undergoing IVF treatment because of male infertility. Human follicular fluid macrophages and luteinized granulosa cells were obtained from pooled follicular fluid of individual patients. Accumulation of VEGF and adrenomedullin in the culture medium of the isolated macrophages and human granulosa cells was determined at variable time intervals ranging from 0 to 48 h. Plasma and follicular fluid concentrations of VEGF and adrenomedullin were also measured. RESULTS: The follicular fluid concentrations of VEGF and adrenomedullin were significantly higher than those found in plasma. After 48 h, accumulation of VEGF in the culture medium of follicular fluid macrophages was significantly higher than that released in the culture medium of luteinized granulosa cells. In contrast, the production rate of adrenomedullin by follicular fluid macrophages was similar to that found in granulosa cells. VEGF secreted by follicular fluid macrophages increased progressively within 48 h of cell culture. A similar response pattern was observed with the culture medium of luteinized granulosa cells, but with lower production rates. CONCLUSIONS: This study suggests for the first time that both luteinized granulosa cells and macrophages actively secrete VEGF and adrenomedullin into follicular fluid in the human ovary.     

The prolactin inhibition of follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells is in part tyrosine kinase and protein kinase-C dependent

The inhibitory actions of prolactin on gonadal steroidogenesishave been reported in different species and under a varietyof experimental approaches. In this study, the mechanisms ofthe in-vitro effects of human prolactin (hPRL) on human folliclestimulating hormone (hFSH)-induced aromatase activity were determinedusing cultured granulosa cells from diethylstilboestrol (DES)-primedimmature rats. Human PRL caused a dose-dependent decrease inhFSH-induced 17ß-oestradiol production, even whencells were cultured in the presence of a cAMP analogue (8-Br-cAMP).These effects of hPRL appeared to be specific, since additionof an anti-rat PRL receptor monoclonal antibody (mAb) mimickedthe hPRL inhibitory effect upon steroidogenesis in rat granulosacells. In order to assess the importance of tyrosine kinaseand protein kinase-C activation in the hPRL inhibitory effectsupon oestrogen biosynthesis, cells were cultured in the presenceof kinase inhibitors. The results showed that addition of genisteinor staurosporine (a tyrosine kinase and protein kinase-C antagonistrespectively) to cultured granulosa cells resulted in potentinhibition of hPRL actions upon hFSH-induced aromatization ina dose-dependent manner. These observations suggest that tyrosinekinase and protein kinase-C activation are involved in the biochemicalevents leading to hPRL inhibitory effects at the gonadal level. ovary/prolactin/protein kinases/steroidogenesis     

Transferrin in the developing ovarian follicle: evidence for de-novo expression by granulosa cells

Transferrin is produced primarily by the liver and is best known as a carrier of iron in the circulation. Transferrin is also produced extra-hepatically where it may serve to suppress the generation of reactive oxygen species and act as a growth factor, in addition to its role in the endocytosis of iron. There is evidence that transferrin and its cognate receptor are important for successful development of follicles but little is known about their precise roles in this context. To learn more about their modus operandi, we undertook immunocytochemical studies which revealed that transferrin and its receptor are distributed heterogeneously in human granulosa cells, with more pronounced expression in more mature follicles. Expression within the oocyte itself was not prominent until the antral stage of development. Using nested reverse trancription-polymerase chain reaction (RT-PCR), transferrin mRNA expression was demonstrated in granulosa cells of the human and mouse ovary but not in the oocyte. Hence it appears that local production of transferrin is possible in addition to the likely uptake of circulating protein into the follicle by endocytosis. Values of transferrin in the follicular fluid were found to be highly correlated with those in serum, suggesting that the small contribution made by its localized synthesis in the granulosa cell may be important for some as yet unknown mechanism in follicle maturation.     

Type 2 11beta-hydroxysteroid dehydrogenase activity in human fallopian tube and correlation of enzyme levels with endometrial histopathology

PROBLEM: The inter-conversion of hormonally active cortisol and inactive cortisone is catalyzed by 11beta-hydroxysteroid dehydrogenase (11beta-HSD). This conversion controls the level of active glucocorticoid concentration in tissues. As the fallopian tube plays a major role in the process of fertilization, we wanted to investigate whether 11beta-HSD is present in the human fallopian tube to control the glucocorticoid levels as in other tissues. METHOD OF STUDY: Isthmic, ampullary and fimbrial portions of the fallopian tube are obtained from patients undergoing hysterectomy and salpingo-oopherectomy for symptomatic leomyomata uteri. 11beta-HSD activities were measured in the homogenates of the tube, cortisol as the steroid substrate. The enzyme activity was expressed as nanomolar cortisone formed per minute per gram of tissue (mean +/- S.D.). RESULTS: A significant level of 11beta-HSD activity in oxidation direction was found in all three parts of the tube. There is no significant difference in the distribution of the enzyme activity throughout the tube. When tubal 11beta-HSD activity was compared with endometrial histology, the enzyme activity is significantly lower in proliferative endometrium when compared with secretory endometrium (P = 0.002). The enzyme activity in inactive endometrium is significantly higher than the active endometrium (P = 0.05). CONCLUSION: The presence of 11beta-HSD throughout the fallopian tube and its correlation to endometrial histology is indicative of its probable role in controlling the glucocorticoid levels in the tissue, which in turn may influence the fertilization process.     

Elimination of apoptotic granulosa cells in atretic follicles: the role of macrophages and intact granulosa cells

The present minireview describes a phenomenon by which phagocytic cells dispose of apoptotic granulosa cells during the process of atresia in mature (graafian) follicles from the guinea pig ovary. 1. Intact granulosa cells take part in the phagocytosis of dying granulosa cells at the early stages of atresia. 2. In addition, macrophages are present within large atretic follicles in order to remove dead granulosa cells and their debris. 3. These two kinds of phagocytic cells remain within the follicle until its atretic stages become considerably advanced. 4. A small number of macrophages are rarely discernible within the growing large follicles of the ovaries during the metestrus period. From these observations, the intrafollicular macrophages and the phagocytic granulosa cells are therefore considered to play an important role in the dynamics of the follicle, especially during both its development and atresia.     

Localization of the progesterone receptor in the porcine ovary

Regulation of progesterone receptor (PR) expression has been studied in many species. However, precise studies have not yet been performed in the porcine ovary. We have examined the localization of PR in follicles and corpora lutea of the porcine ovary at different stages of their development. The effects of LH and FSH on PR expression in granulosa cells of small antral follicles was also studied. Immunohistochemistry was applied to determine the distribution of PR while immunoblot analysis showed that two isoforms A and B were present. Early antral follicles contained PR in the granulosa layer. In granulosa cells of small and medium antral follicles PR was not detected whereas it was present in the theca layer. Before ovulation, PR was found in both granulosa and theca cells of large follicles and the staining intensity was very strong. FSH or LH treatment of small follicles (100 ng/ml) induced changes in cellular distribution patterns of PR. In both cases, PR was expressed in granulosa cells. PR was detected in corpora lutea in all 3 stages of the luteal phase. Our data show that in the pig ovary changes in PR localization are stage-specific and suggest that expression of PR is positively regulated by both LH and FSH.     

The transient disappearance of cytokeratin in human fetal and adult ovaries

Cells from the inner and outer granulosa cell layers of the ovarian follicles differ in function, probably because of their different origins from the surface epithelium and from the rete. This suggestion has not so far been thoroughly investigated in the human ovary. We examined fetal ovaries from the early, middle and late gestational periods, ovaries from fertile women, and preovulatory follicular cells obtained from patients under in vitro fertilization therapy (IVF). Indirect immunohistology and immunocytology were used to detect the presence of cytokeratin (CK)-positive epithelial cells. In fetal ovaries from the early gestational period, prominent rete tubules (sometimes with oocytes) appeared to be fused with the sex cords and primordial follicles. Both showed CK-positively, detected with the pan-CK antibody Lu-5. Cytokeratin 19 was clearly expressed in the fusion area. In the fetal and adult ovaries, CK-positive follicular or granulosa cells were noted in the primordial and primary follicles as well as the preovulatory follicles. Cytokeratin was not detected in the granulosa cells of growing follicles, CK-positive and -negative luteal cells were identified in the developing corpus luteum. We conclude for the human ovary: (1) the heterogeneous morphology of granulosa cells may be explained by their twofold origin from the surface epithelium and the rete, (2) the rete tubules appear to be involved in folliculogenesis, (3) the transient absence of CK expression in growing follicles compared to resting and mature follicles or to the developing corpus luteum indicates a particular role of CK-positive cells at the periovulatory period. Accepted: 20 September 1999     

11Beta-hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction

The type 2 isoform of 11beta-hydroxysteroid dehydrogenase (11beta- HSD2), which inactivates cortisol (F) to cortisone (E), has been suggested to play a role in the ontogeny of the fetal pituitary-adrenal axis and also protect the developing fetus from the deleterious effects of circulating maternal glucocorticoids. The abundance of 11beta-HSD2 in the placenta and other fetal tissues was inferred from the F/E ratio in 17 term deliveries in both umbilical arterial (1.73 +/- 0.24, mean +/- SE) and umbilical venous blood (1.16 +/- 0.14) compared with adult peripheral venous blood (7.76 +/- 0.57, n = 70). Using sensitive assays for 11beta-HSD2 and an in-house human 11beta-HSD2 antibody, the expression and activity of this enzyme in fresh frozen human placenta increased progressively from first (8-12 weeks, n = 16) and second (13- 20 weeks, n = 9) to third trimester (term) pregnancies (39-40 weeks, n = 50). Placental 11beta-HSD2 activity was significantly reduced in deliveries complicated by intrauterine growth restriction (IUGR) [25-36 weeks, n = 12, activity 380 pmol/mg/h median (225-671; 95% confidence interval)], compared with the term deliveries [888 (725-1362)] and with appropriately grown pre-term deliveries [27-36 weeks, n = 14, activity 810 (585-1269)], P < 0.05. In human pregnancy placental 11beta-HSD2 activity increases markedly in the third trimester of pregnancy at a time when maternal circulating levels of glucocorticoid are rising. The finding of attenuated placental 11beta-HSD2 activity in IUGR suggests that glucocorticoids may, in part, contribute to impaired fetal growth and that this is closely controlled in normal gestation through placental 11beta-HSD2 expression.      

Bone Morphogenetic Protein‐2 (BMP‐2) Increases Gene Expression of FSH Receptor and Aromatase and Decreases Gene Expression of LH Receptor and StAR in Human Granulosa Cells

Citation Shi J, Yoshino O, Osuga Y, Koga K, Hirota Y, Nose E, Nishii O, Yano T, Taketani Y. Bone morphogenetic protein‐2 (BMP‐2) increases gene expression of FSH receptor and aromatase and decreases gene expression of LH receptor and stAR in human granulosa Cells. Am J Reprod Immunol 2011; 65: 421–427 Problem A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP‐2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP‐2 in the human ovary. Method of Study BMP‐2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP‐2 or human chorionic gonadotrophin (HCG), followed by RNA extraction. Results BMP‐2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP‐2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP‐2 and increased BMP and activin membrane‐bound inhibitor (BAMBI), an antagonist of BMP‐2. Conclusion Expression and disappearance of BMP‐2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP‐2 function by controlling BMP‐2 and BAMBI expression.     

11beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone stimulates chorionic gonadotropin secretion from human term cytotrophoblast cells differentiated in vitro

PROBLEM: To investigate the effect of altering local glucocorticoid concentration on human chorionic gonadotropin (hCG) production by cultured placental trophoblast cells. METHOD OF STUDY: Human placental trophoblasts were isolated from fresh placentas. Cytotrophoblasts were purified and placed into 24-well multiplates. For cultivation Dulbecco's modified Eagle's medium (DMEM) with 15 mm HEPES and 15% FBS was used. 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) activity and its inhibition by carbenoxolone (CE) were measured in cultured cells. Cultures were exposed to CE for 16-20 hr. Overnight production of hCG was measured by radioimmunoassay in control and treated cells. RESULTS: The 11beta-HSD activity in these cultures was inhibited by nm concentrations of CE, the apparent Ki being 2.5 nm. Inhibition of 11beta-HSD activity with 0.1 nm CE resulted in 1.5-fold increase in the production of hCG. CONCLUSIONS: Increasing local glucocorticoid concentration by the inhibition of 11beta-HSD results in higher hCG secretion, which in turn enhance cell differentiation.     

Cultured human granulosa cells secrete a follicle stimulating hormone receptor-binding inhibitor

We previously reported that human follicular fluid contained a protein that inhibits binding of 125I-human FSH to its membrane receptor (FSH- BI) and demonstrates FSH-like agonist activity in vitro. The cellular origin of FSH-BI was unknown, although ovarian granulosa cells seemed a likely source. To address this question, human granulosa cells were collected from patients during routine in-vitro fertilization (IVF) or gamete intra-Fallopian transfer (GIFT) procedures. Cells from 98 patients were cultured and then examined for their ability to secrete FSH receptor-binding inhibitory activity into the culture medium. The function of the cultured cells was confirmed by their ability to respond to added FSH with conversion of exogenous androstenedione to oestradiol. Radioreceptor assays were performed individually on cell culture medium obtained from granulosa cell cultures from these 98 patients. Cultured granulosa cells, under basal conditions (in the absence of FSH stimulation), secreted significant FSH-BI activity into the culture medium. In order to accumulate enough material for further study, this culture medium was pooled and lyophilized. The lyophilized medium retained FSH-BI activity, and also demonstrated FSH agonist activity by stimulating oestradiol synthesis in cultured rat Sertoli cells. A fraction showing a single component after purification by polyacrylamide gel electrophoresis had an estimated molecular weight of 25000, and inhibited 125I-human FSH binding to receptor by 50% at 2.5 microg/ml. The results indicate that human granulosa cells secrete a protein with FSH-like activity having potential significance as a local regulator of FSH action in the ovary.      

Transforming growth factor-beta isoforms and receptors in endometriotic cysts of the human ovary.

PROBLEM: The present study examined the presence and cellular distribution of transforming growth factor-beta1, 2, and 3 isoforms and their type I and II receptors in endometriotic cysts of the ovary, relative to their presence in normal endometrial tissue. METHOD OF STUDY: Thirteen control samples of normal endometrium in the proliferative phase and 11 ovarian endometriotic cysts were examined by immunohistochemistry for transforming growth factor-beta1, 2, and 3 isoforms and their type I and II receptors. RESULTS: Immunoreactivity for all ligands and receptors was detected in both normal endometrium and endometriotic cysts. Isoform-specific differences in immunostaining were not detected. Expression of all ligands and receptors was significantly increased in epithelial cells of endometriotic cysts compared with those of normal endometrium. On the other hand, stromal cells in normal endometrium and endometriotic cysts were only faintly immunostained. Inflammatory cells infiltrating among endometriotic stromal cells contained the highest immunostaining intensity for all ligands and receptors. We identified nearly all inflammatory cells as macrophages using a specific antibody. CONCLUSIONS: These findings suggest that macrophages in endometriotic tissue are a major source of transforming growth factor-beta, which may be an important regulator of cell proliferation in endometriotic cysts through paracrine and autocrine actions.