Altered expression of activin, cripto, and follistatin in the endometrium of women with endometrioma

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1 Altered expression of activin, cripto, and follistatin in the endometrium of women with endometrioma Ana Luiza L. Rocha, M.D., a Patrizia Carrarelli, B.Sc., a Romina Novembri, B.Sc., a Lorenzo Sabbioni, M.D., a Stefano Luisi, M.D., Ph.D., a Fernando M. Reis, M.D., Ph.D., b and Felice Petraglia, M.D. a a Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy; and b Department of Obstetrics and Gynecology, Federal University of Minas Gerais, Belo Horizonte, Brazil Objective: To evaluate the expression pattern of activin A, activin receptors, and activin modulators messenger RNA (mrna) in the eutopic endometrium of patients with endometriosis at different phases of the menstrual cycle and to evaluate the mrna expression of the same proteins in endometriomas during the menstrual cycle. Design: Prospective study. Setting: University hospital. Patient(s): Women with and without endometriosis. Intervention(s): Samples of endometrial and endometriotic tissue from women with endometrioma (n ¼ 48), and endometrial samples from women without endometriosis (controls) (n ¼ 48). Main Outcome Measure(s): Quantification of activin A, activin B, activin receptor II, nodal, cripto, inhibin a, and follistatin expression by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Result(s): The eutopic endometrium of patients with endometriosis showed [1] higher activin A mrna expression in the proliferative phase and a lack of late secretory phase peak, [2] a lack of endometrial cycle-related variations of cripto and inhibin a mrna expression, and [3] an inverse expression pattern of follistatin mrna. Endometriomas showed similar variations in the expression of activin-related protein mrna during the menstrual cycle as eutopic endometrium. Conclusion(s): The disturbed expression of endometrial activin A, cripto (activin receptor antagonist), and follistatin (activin-binding protein) suggests a dysfunction of the activin pathway in endometriosis. Endometriomas showed similar changes of activin-related proteins during the menstrual cycle, which supports a common biology for eutopic and ectopic endometrium in endometriosis. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Activin A, activin B, activin receptors, cripto, endometriosis, follistatin, inhibin a, nodal Endometriosis is a pathologic gynecologic entity typical of women in reproductive age, associated with pelvic pain and infertility (1). The disease can manifest in three clinically distinct forms: pelvic endometriotic implants, ovarian endometriomas, and deep endometriotic nodules (1, 2). These variants of the same disease have in common histologic features as chronic bleeding and signs of inflammation (1, 2). Endometriotic tissue proliferates in the peritoneal environment and maintains a histologic similarity to eutopic endometrium (2). Indeed, ectopic implants seem to remain functionally responsive to sex steroid hormones but with differences in their protein production and receptor expression (3). Endometriotic cysts express matrix metalloproteinases (MMP) and angiogenic factors in higher amounts with respect to eutopic endometrium, suggesting that the endometrial tissue undergoes modifications that enable its implantation in the peritoneal cavity and pelvic organs (4). Moreover, Received November 10, 2010; revised March 1, 2011; accepted March 11, 2011; published online April 15, A.L.L.R. has nothing to disclose. P.C. has nothing to disclose. R.N. has nothing to disclose. L.S. has nothing to disclose. S.L. has nothing to disclose. F.M.R. has nothing to disclose. F.P. has nothing to disclose. Supported by the University of Siena, Italy; Coordenaç~ao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and Brazilian National Institute of Hormones and Women s Health. Reprint requests: Felice Petraglia, M.D., Obstetrics and Gynecology, Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico Santa Maria alle Scotte, Viale Bracci, Siena, Italy ( petraglia@unisi.it). ectopic endometrium has a decreased expression of relaxin and urocortin messenger RNA (mrna) expression, which supports a difference with eutopic tissue (5, 6). Expression of estrogen and progesterone receptors is quantitatively altered in endometriotic tissue and biologically significant quantities of progesterone and estrogen are produced locally in endometriotic tissue through an abnormally active steroidogenic cascade (2). Molecular and biochemical studies also have revealed that the eutopic endometrium of patients with endometriosis shows differences in comparison with healthy women, which suggests a role of endometrial dysfunction in infertility (7 9); avb integrins in particular are reduced at the time of implantation (7, 10). Transforming growth factor b (TGF-b) and its family members are expressed by human endometrium, acting on cell proliferation, differentiation, immune function, apoptosis, and tissue remodeling and playing a role in the menstrual cycle, decidualization, and early pregnancy (11, 12). The TGF-b superfamily includes activins and inhibins, which are closely related dimeric glycoproteins (13, 14). Activin A (ba ba) is produced by the endometrium (15, 16), with the highest mrna expression in the secretory phase (17) under progesterone andinterleukin1(il-1)modulation(18); it plays a possible role in the process of decidualization (19, 20). Activin B (bb bb) is also expressed by the endometrium (16), is related to the degree of endometrial decidualization, and is reduced in tubal ectopic pregnancy (21). The biologic effects of activins are mediated by specific receptors (ActR), encoded by different genes, ActRI and ActRII, that initiate the Smad phosphorylation cascade (22, 23) and are localized in human /$36.00 Fertility and Sterility â Vol. 95, No. 7, June doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 TABLE 1 Baseline characteristics of two groups, healthy controls and women with endometriosis, for different phases of the menstrual cycle. Control Endometriosis Characteristic EP LP ES LS EP LP ES LS Total Patients, n Mean age (y) Standard deviation (5.6) (5.8) (6.4) (5.9) (4.3) (4.0) (6.5) (4.0) (5.3) Note: EP ¼ early proliferative (days 5 to 10); LP ¼ late proliferative (days 11 to 14); ES ¼ early secretory (days 14 to 22); LS ¼ late secretory (day 23 onward). endometrium (24). Nodal and cripto are ActRs modulators. Nodal is a member of the TGF-b superfamily that requires the presence of a coreceptor, cripto, to bind ActR (25). Nodal regulates the processes of formation and differentiation that occur during early embryo development (26) and its pathway activity is up-regulated in many human cancers (27). Cripto is a cell membrane glycosylphosphatidylinositolanchored glycoprotein that functions as a coreceptor for activin by forming a complex with activin receptors and antagonizing its effects (28). Cripto has the ability to initiate several aspects of tumor progression, including increased proliferation, migration, invasion, angiogenesis, and epithelial-to-mesenchymal transition (29). Cripto is highly expressed in endometrial stromal and epithelial cells during the proliferative phase of the menstrual cycle, and its expression is up-regulated in endometrial cancer (30). Inhibins antagonize the actions of activin through competitive binding to ActRII, nodal, and cripto (25, 28). Inhibin a has been primarily detected in the glandular and luminal epithelial cells of human endometrium and has shown an increased expression in the secretory phase (31). Inhibin a is up-regulated in decidualized stromal cells, during the secretory phase, and also after administration of exogenous progestins, which implies a putative regulation by progesterone and suggests a role of this protein in human embryo implantation (16, 32). The actions of activin A are also modulated by follistatin, an activin-binding protein, by binding to activin with high affinity and blocking its interaction with the receptor (33). Follistatin also binds and modulates the actions of several other members of the TGF-b family such as myostatin and certain bone morphogenetic proteins, playing a critical role in several tissues (33, 34) and in human endometrium (17, 24, 35). The evidence that endometriotic tissues express activin A (36),ActRII, nodal, cripto (37), and follistatin (38) and that activin A increases the invasiveness of endometrial cells in an in vitro model of human peritoneum suggest a possible role of activin pathway in the development of endometriosis (39). We evaluated whether in endometriotic women the endometrial mrna expression of activin A, activin B, ActRII, inhibin a, cripto, and nodal follistatin throughout the menstrual cycle differs from healthy women. In addition, the same mrna levels were analyzed in endometriomas to evaluate whether the ectopic tissues would show the same changes as eutopic endometrium. MATERIALS AND METHODS Endometrial Sample Collection Samples of human endometrium were obtained from nonpregnant patients undergoing gynecologic surgery by laparoscopy (n ¼ 96). All women were between 21 and 39 years of age ( years, mean standard deviation [SD]) and had normal and regular menstrual cycles. The study population consisted of two groups of women: group 1 was patients with endometrioma who were undergoing laparoscopic treatment (n ¼ 48); group 2 was healthy women (n ¼ 48) who were undergoing laparoscopy for tubal sterilization (controls). In both groups, the proliferative phase samples were separated into early proliferative (days 5 to 10) and late proliferative (days 11 to 14); the secretory phase samples were separated into early (days 14 to 22) and late (day 23 onward) (Table 1). Pieces of endometrial tissue (approximately 1 cm 3 ) were obtained from women with regular menstrual cycles (28 to 30 days). In the same group of patients, a sample of endometriotic tissue was collected from ovarian endometriomas during surgical intervention. The endometriotic tissue samples were carefully stripped from the cyst wall lined by endometriotic tissue without taking any of the normal ovarian cortex, as confirmed by histologic evaluation (40). Eutopic endometrium was collected by hysteroscopy, and all samples were classified according to the last day of menstruation, as confirmed by a transvaginal ultrasound scan (41) and by histologic criteria. In group 1, the cyst diameter measured by ultrasound ranged from 38 to 72 mm, and all patients included had stage III or IVendometriosis according to the American Society for Reproductive Medicine classification (42). They were in treatment in our infertility center to achieve pregnancy; other endocrine/uterine or male factors were excluded. A complete medical history was obtained from each woman, and a physical examination was performed. Patients who had undergone hormone treatment in the last 3 months were excluded from the study. All specimens were immediately submerged in liquid nitrogen to allow subsequent RNA extraction and real-time polymerase chain reaction (PCR). All study participants gave their written informed consent before participating in this study, which was approved by the local human investigations committee. RNA Extraction and cdna Preparation Samples were disrupted and homogenized by passing the lysate at least five times through a blunt 20-gauge needle (0.9 mm diameter) fitted to an RNAsefree syringe. The total RNA was extracted with the RNeasy Protect Mini Kit and then treated with RNase-free DNase according to the manufacturer s instructions (RNase Protect Mini Kit; Qiagen, Hilden, Germany). The RNA was quantified by ultraviolet absorption (OD260), and 300 mg were reverse-transcribed using the High Capacity cdna Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-Time Polymerase Chain Reaction Endometrial cycle-related changes in the eutopic and ectopic endometrium of the mrna expression of activin A, activin B, and activin receptor modulators and antagonists were compared by RT-PCR. The RT-PCR was run in an Opticon 2 thermal cycler (MJ Research, Bio-Rad Laboratories, Waltham, MA) using TaqMan gene expression assays (Applied Biosystems, 2242 Rocha et al. Activin and endometriosis Vol. 95, No. 7, June 2011

3 Weiterstadt, Germany). The housekeeping gene 18S ribosomal RNA (assay identification no. Hs _m1) was used as the internal control. All samples were run in triplicate in 96-well optical PCR plates (Applied Biosystems), optimized to the universal PCR protocol of the manufacturer with a TaqMan Universal PCR Master Mix (Applied Biosystems). Commercially available TaqMan assays (Applied Biosystems) were used for activin A (assay identification no. Hs _m1), activin B (assay identification no. Hs _m1), ActRII (assay identification no. Hs _m1), cripto (assay identification no. Hs _m1), nodal (assay identification no. Hs _m1), inhibin a (assay identification no. Hs _m1), and follistatin (assay identification no. Hs _m1). A standard complementary DNA (cdna) sample prepared from midsecretory endometrium was diluted serially to construct the relative standard curves that were used to quantify the PCR results. The threshold cycle for amplification of each sample was used to calculate its input amount of target cdna through the linear equation generated by the standard curve. The results are given as the ratio between the calculated input amount of each gene and that of 18S, expressed in arbitrary units (43). The following thermal cycle protocol was applied: after initial denaturation for 10 minutes at 95 C, denaturation at the subsequent 45 cycles was performed for 15 seconds at 95 C, followed by primer annealing and elongation at 60 C for 1 minute. Blank samples for each reaction consisted of amplifications performed in the absence of the RT enzyme. FIGURE 1 (A) Activin A, (B) activin B, and (C) ActRII messenger RNA (mrna) expression in eutopic endometrium of healthy patients (controls) and women with endometriosis and in endometriotic tissue (endometrioma) during the proliferative and secretory phases of the menstrual cycle *P<.05; **P<.01; ***P<.001; P<.05 versus control. EP ¼ early proliferative; LP ¼ late proliferative; ES ¼ early secretory; LS ¼ late secretory. Statistical Analysis Data distribution was evaluated by the Kolmorov-Smirnov normality test with Prism 4 computer software (GraphPad Software, La Jolla, CA). After the normality test confirmed that the values did not depart significantly from the normal curve, the statistical significance of the differences found between the four menstrual cycle phases was assessed using one-way analysis of variance (ANOVA) followed by the post hoc Tukey s test for multiple comparisons. Two group comparisons (control group vs. women with endometriosis) were performed with an unpaired Student s t-test. The PCR results are plotted as mean standard error. P<.05 was considered statistically significant. RESULTS Eutopic Endometrium Activin A, activin B, activin receptor, follistatin, nodal, cripto, inhibin a, and follistatin mrnas were expressed in all eutopic endometrium samples evaluated. Gene expression analysis was performed in all phases of the endometrial cycle in healthy women and in patients with endometriosis; in each group of patients, the gene expression variation throughout the cycle was evaluated. Moreover, each menstrual phase was compared between controls and endometriotic patients. Activin A, activin B, and activin receptor Activin A mrna expression in healthy controls in the late secretory phase was statistically significantly higher than in the proliferative phase (P<.001), but no changes were observed in patients with endometriosis along the menstrual cycle (Fig. 1A). However, an up-regulated expression in the early and late proliferative phase was observed (P<.05) in patients with endometriosis compared with controls. Activin B mrna expression did not show any statistically significant differences between the proliferative and secretory phases in healthy women or in patients with endometriosis (see Fig. 1B). No differences in ActRII mrna expression were found (see Fig. 1C). Activin receptor modulators: nodal and cripto Nodal mrna expression did not show any statistically significant differences between women with endometriosis and healthy controls in either the proliferative or secretory phases (Fig. 2A). In the healthy controls, expression of cripto mrna was markedly higher during the Fertility and Sterility â 2243

4 FIGURE 2 (A) Nodal and (B) cripto messenger RNA (mrna) expression in eutopic endometrium of healthy patients (controls) and women with endometriosis and in ectopic tissue (endometrioma), during the proliferative and secretory phases of the menstrual cycle. *P<.05; ***P<.001; versus control. EP ¼ early proliferative; LP ¼ late proliferative; ES ¼ early secretory; LS ¼ late secretory. FIGURE 3 (A) Inhibin a and (B) follistatin messenger RNA (mrna) expression in eutopic endometrium of healthy patients (controls) and women with endometriosis and in ectopic tissue (endometrioma) during the proliferative and secretory phases of the menstrual cycle. *P<.05. EP ¼ early proliferative; LP ¼ late proliferative; ES ¼ early secretory; LS ¼ late secretory. proliferative phase (P<.001) than during the secretory phase. In patients with endometriosis, cripto was down-regulated (P<.05), and no difference was found between the proliferative and secretory phases of the endometrial cycle (see Fig. 2B). Activin antagonist and binding protein: inhibin a and follistatin Inhibin a mrna expression differed between the patients with endometriosis and the healthy controls. The healthy controls exhibited an increased expression of inhibin a mrna during the secretory phase (P<.05 vs. the proliferative phase). In patients with endometriosis, no difference in expression was found between the proliferative and secretory phase samples (Fig. 3A). Follistatin mrna expression in eutopic endometrium did not show any statistically significant difference between the proliferative and secretory phases in healthy controls. Conversely, in women with endometriosis the follistatin mrna expression during the secretory phase was higher (P<.05) than during the proliferative phase (see Fig. 3B). No differences of follistatin mrna expression were found in the eutopic endometrium of the controls or the women with endometriosis. Ectopic Tissue Activin-related protein mrna was evaluated and found to be expressed in the endometriomas of patients with endometriosis. Similar to the eutopic endometrium of women with endometriosis, the activin A, activin B, activin receptor, nodal, cripto, and inhibin a mrna expression in endometriomas did not change between the proliferative and secretory phases (see Figs. 1, 2, and 3A); follistatin mrna expression was higher during the secretory phase (P<.05 vs. proliferative phase) (see Fig. 3B) Rocha et al. Activin and endometriosis Vol. 95, No. 7, June 2011

5 DISCUSSION Our study has shown that the eutopic endometrium has a different menstrual cycle-related pattern of activin regulation in patients with endometriosis when compared with healthy controls. In particular, activin A, cripto, and inhibin a mrna expression did not change throughout the menstrual cycle, and we found an increase of follistatin mrna during the late secretory phase. Moreover, our study also revealed that endometriomas show the same variations during the endometrial cycle as the eutopic endometrium. Several mechanisms have been proposed to explain the association between endometriosis and infertility, including distorted pelvic anatomy, endocrine and ovulatory abnormalities, and altered hormone and immune cell-mediated functions in the endometrium (10, 44). The behavior of endometriotic implants may affect embryo implantation (10, 45), but evidence suggests that endometrial dysfunction also occurs (7, 10). Delayed histologic maturation and biochemical disturbances support a role for endometrial dysfunction as a contributor to a decrease of fertility in women with endometriosis (8, 10, 45). Indeed, endometrial corticotropin-releasing hormone (CRH) and urocortin peptides involved in endometrial decidualization were down-regulated in endometriotic patients (6). Steroid hormones stimulate local expression and synthesis of several proteins that are up-regulated or induced throughout the endometrial cycle phases and in early pregnancy. These locally expressed peptides/proteins participate in paracrine signaling to other cell types to modulate endometrial functions; their dysfunction may initiate pathologic conditions such as infertility in endometriosis. In our study, the women with endometriosis showed no increase of activin A mrna expression during the secretory phase. The high expression of activin A mrna during the secretory phase of healthy women (17) has been suggested to be a signal of a receptive endometrium (17, 18), which may promote trophoblast invasion during embryo implantation (46). Therefore, the lack of any secretory change in endometriotic patients may be related to an infertile endometrium. In addition, activin A is active on neovascularization (47), increases atrix metalloproteinase (MMP) activity (48), and is involved in the infiltration of macrophages through the basement membrane in an inflammatory state (49), supporting several possible sites of activin action in the pathogenesis of endometriosis. Indeed, a recent study showed that activin A increased the ability of endometrial cells to invade peritoneum in vitro (39). The expression of cripto mrna in patients with endometriosis did not change during the endometrial cycle, missing the proliferative phase peak exhibited by healthy controls. Therefore, in patients with endometriosis an enhanced activin effect also may occur because of a reduced antagonist expression during the proliferative phase, probably causing a loss of control of cell proliferation (16, 17). Inhibin a mrna expression did not change in the patients with endometriosis, but the control women had a higher endometrial expression of the a subunit during the late secretory phase, in agreement with previous studies (16, 21, 32, 42, 50, 51). The lack of inhibin a expression in endometriotic women during the secretory phase may further contribute to an impaired endometrial function in these patients. The evidence that patients with endometriosis showed increased endometrial follistatin mrna expression during the secretory phase may also contribute to a reduced activin effect on endometrial functions in endometriosis. Moreover, follistatin induces angiogenesis and is up-regulated during endothelial cell proliferation and migration (52), facilitating the invasion and proliferation of endometriotic cells. To a certain extent, follistatin up-regulation can be considered a promoter of the endometriotic lesion genesis once it stimulates angiogenesis and cell proliferation (35). Our study showed that the secretory phase eutopic endometrium of women with endometriosis is characterized by decreased inhibin a and increased follistatin mrna expression in the presence of impaired activin A expression, which explains in part the impaired decidualization and possibly affects embryo implantation (7, 10, 45). Abnormalities in the endometrium that result in embryo implantation failure are largely believed to be involved in the lower pregnancy rates of women with endometriosis (8). Some differences were observed in the eutopic endometrium of patients with endometriosis in comparison with healthy women, in particular reduced avb integrins at the time of implantation (7, 10); a very low level of an enzyme involved in the synthesis of endometrial ligand for L-section (a protein present at trophoblast blastocyst interface) (10); and the genes for apoptosis, ion transporters, immune functions, secretory proteins, signal transduction, membrane proteins, transcription factors, and others that likely are relevant in implantation failure and the pathogenesis of the disease (8). Furthermore, our study demonstrated that endometriomas show the same variations of the eutopic endometrium during the menstrual cycle. This suggests a common biology for eutopic and ectopic endometrium in endometriosis, but does not verify whether these changes in eutopic endometrium are secondary to the development of endometriotic lesions or are primary defects that remain in the ectopic tissue. On the other hand, other studies have shown differences between ectopic and eutopic tissue in endometriosis. The levels of progesterone receptor are significantly decreased, and there is a pathologic overexpression of ERb in endometriotic stromal cells (2, 53). In endometriotic tissue, there is an abnormally active steroidogenic cascade, and significant quantities of progesterone and estrogen are produced locally (2, 54, 55). Proliferative and secretory endometrium in patients with endometriosis is characterized by impaired activin A, cripto, inhibin a, and follistatin mrna expression, demonstrating that eutopic endometrium is distinct between endometriotic patients and healthy women. The endometriomas showed the same changes of eutopic endometrium during the endometrial cycle, which supports the existence of a similar biological activity between these two endometrial tissues. 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