ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis Patrizia Carrarelli, Ph.D.,a Chih-Fen Yen, M.D.,b,c Felice Arcuri, Ph.D.,a Lucia Funghi, Ph.D.,a Claudia Tosti, M.D.,a Tzu-Hao Wang, M.D.,b,d Joseph S. Huang, M.D., Ph.D.,e and Felice Petraglia, M.D.a a Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; b Department of Obstetrics and Gynaecology, Chang Gung Memorial Hospital at Linkou and Chang Gung University College of Medicine, Taoyuan, Taiwan; c Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Taoyuan, Taiwan; d Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan; and e Department of Obstetrics and Gynaecology, The Ohio State University College of Medicine, Columbus, Ohio
Objective: To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. Design: Prospective study. Setting: University hospital. Patient(s): Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. Intervention(s): Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. Main Outcome Measure(s): Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-a, cripto), and receptors (ActRIIa, ActRIIb) were performed. Result(s): Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-a, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. Conclusion(s): The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility. (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive Use your smartphone Medicine.) to scan this QR code Key Words: Adenomyosis, nodular adenomyosis, eutopic endometrium, activin, inhibin, and connect to the myostatin, follistatin, activin receptors, crypto, infertility Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/carrarellip-activin-growth-factors-adenomyosis/
A
denomyosis is a benign uterine disorder, clinically characterized by abnormal uterine
bleeding, menorrhagia, dysmenorrheal, and infertility. Its main pathologic feature is the presence of endometrial
Received March 10, 2015; revised May 13, 2015; accepted May 24, 2015. P.C. has nothing to disclose. C.-F.Y. has nothing to disclose. F.A. has nothing to disclose. L.F. has nothing to disclose. C.T. has nothing to disclose. T.-H.W. has nothing to disclose. J.S.H. has nothing to disclose. F.P. has nothing to disclose. Patrizia Carrarelli and Chih-Fen Yen have a similar in author order. Supported by Chang Gung Memorial Hospital research grants CMRPG3C0672 to C.-F.Y. Reprint requests: Felice Petraglia, M.D., Obstetrics and Gynaecology, Department of Molecular and Developmental Medicine, University of Siena, Policlinico ‘‘Santa Maria alle Scotte,’’ Viale Bracci, 53100 Siena, Italy (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.032 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
glands and stroma diffuse within myometrium, or circumscribed in nodular aggregates of endometrial glands and stroma surrounded by myometrial hyperplasia and hypertrophy (adenomyoma) (1, 2). Clinically, endometriosis and uterine leiomyoma share similar clinical symptoms and some diagnostic features with adenomyosis (3). Adenomyosis is known to be a hormone-related disorder, characterized by high E2 receptor and impaired 1
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY P receptor expression (4, 5). Estrogens (Es) are essential for the development of adenomyosis, as they enhance the growth of endometrial glands and stroma into myometrium (1) as well as myometrial growth (6). In addition, E-associated epithelial-mesenchymal transition plays a potential role in the invasive features of endometrial cells in adenomyosis (7), with local E production induced by increased aromatase and E sulfatase activities in adenomyoma (1, 8). The epithelial-mesenchymal transition is a process that allows epithelial cells to detach from neighbor cells, to transverse the dissolving basement membranes, and to move to the extracellular matrix to reach other tissues. This process takes place during development, but it is also the mechanism used by cancer cells to colonize contiguous tissues (9). It is one of the most widely accepted proposed mechanisms underlying the myometrial invasion of the endometrium in adenomyosis. In an adenomyosis model, tissue growth, invasion, and angiogenesis occur through an annexin A2-induced E-dependent epithelial-mesenchymal transition (10). It has been proposed that, in specific cases, adenomyosis may originate from the invagination of endometrial cells into a weakened myometrium after a surgical lesion or as consequence of physiologic conditions (such as increased intrauterine pressure during pregnancy) (1, 11) or by myometrial dysfunction (1, 12). Adenomyosis is also considered an endometriosis-like disease, as endometrial invasion of myometrium underlies some of the pathogenic mechanisms of peritoneal endometriosis (i.e., cell proliferation, invasion, and neovascularization), associated with inflammation and tissue remodeling (13). Like endometriosis, adenomyosis is associated with the presence of a more invasive endometrium (14, 15). Therefore, the myometrial components may have a role in the etiology of adenomyosis as invasion is facilitated by the loss of cohesion of myometrial bundles influenced by matrix metalloproteinases (MMPs) (14, 16). Regarding molecular aspects of the pathogenesis of adenomyosis, previous studies from our and other groups have shown a deregulation of genes involved in inflammation, protease activation, and autophagy. In particular, molecules involved in extracellular matrix remodeling (MMP-2 and MMP-9) and angiogenesis, like vascular endothelial growth factor and microvessel density, are hyperexpressed in adenomyosis (17, 18). In addition, eutopic and ectopic endometria of adenomyosis showed increased interleukin expression, leading to impaired fertility in these patients (19). The transforming growth factor beta (TGF-b) superfamily includes activins, bone morphogenetic proteins, growth differentiation factors, and myostatin. Activin A (bA-subunits dimer) mediates a wide range of biological activities including wound healing, cell proliferation and differentiation, immune response, and angiogenesis (20, 21). In target cells, the binding of this growth factor to its type II receptor (ActRIIa or ActRIIb) leads to the recruitment, phosphorylation, and activation of specific type I receptor, also known as activin receptor-like kinase. Intracellular signaling is subtly regulated by cell-specific and context-dependent inactivating mechanisms by several membrane (cripto) and extracellular 2
receptor (inhibins) antagonists, and the activin-binding protein follistatin (22). In humans endometrium activin A plays a role in the menstrual cycle, with higher expression during the late secretory phase, and decidualization (23, 24). Furthermore, activin A messenger RNA (mRNA) and protein expression are significantly increased in endometrial adenocarcinoma (25), as well as in the serum and uterine washing fluid of patients affected by endometrial cancer (26). Activin A modulates the expression and secretion of interleukin-8 and vascular endothelial growth factor in cultured human endometrial stromal cells, and this mechanism is disrupted in eutopic endometrial cells from women with endometriosis (27). In addition, activin A increases endometrial cells invasiveness in an in vitro model of peritoneum, suggesting its possible role in the pathogenesis of endometriosis (28). Myostatin is another member of the TGF-b superfamily, and is a negative regulator of muscle size (29). Enlargement of the muscle mass is observed when myostatin signaling is impaired in transgenic mice carrying a dominant negative form of the myostatin receptor or in transgenic mice overexpressing follistatin, which binds and inhibits myostatin (29). Myostatin and activin A regulate myometrial cell proliferation (30, 31) and their expression is increased in uterine leiomyoma (32). Furthermore, activin A and myostatin significantly affect fibronectin, collagen1A1, and versican in primary myometrial cells and leiomyoma cells (33). Follistatin is a binding protein that stimulates muscle growth by direct inhibition of myostatin (29) and is involved in the inflammatory response by inhibiting activins and bone morphogenetic protein signaling, and is up-regulated during endothelial cell proliferation and migration (34). Follistatin expression is aberrant in endometrioma (35) and in eutopic endometrium of women with endometriosis (36), suggesting that dysfunction of this pathway may contribute to the infertility of patients with endometriosis. To our knowledge, no studies have investigated follistatin expression in adenomyosis. The present study aimed to investigate the mRNA expression of activin-related growth factors in adenomyotic tissues (nodules) and in eutopic endometrium compared with endometrium of control women. There is a possible role of these growth factors in the pathogenesis of adenomyosis.
MATERIALS AND METHODS Tissue Collection Endometrium and adenomyotic tissues (nodules) were obtained from symptomatic women, who were not pregnant and undergoing hysterectomy (age range, 38–42 years). The Institutional Review boards of Chang Gung Memorial Hospital and of the University of Siena approved this study. A written informed consent was obtained from each woman. The patient's medical history was recorded and a complete physical examination was performed based on reports of symptoms of dysmenorrhea, menorrhagia, abnormal bleeding, and pelvic pain. VOL. - NO. - / - 2015
Fertility and Sterility® The study population consisted of two groups of women: [1] patients with nodular adenomyosis (adenomyoma) (n ¼ 8) undergoing hysterectomy; and [2] control women (n ¼ 12) undergoing hysterectomy for prolapse. The diagnosis of nodular adenomyosis was carried out by two-dimensional and power Doppler pelvic and vaginal ultrasound (37). Exclusion criteria were patients receiving hormonal treatment at least 1 month before surgery, pregnancy, menopausal status, and reproductive tract cancer. Patients were excluded if preoperative ultrasound examination revealed submucous or intramural myomas (>3 or >4 cm). Adenomyotic nodules (diameter, 8–20 mm) were dissected immediately after hysterectomy, followed by histologic confirmation by a pathologist. Specimens of eutopic endometrium and from control women were collected by hysteroscopy. All surgeries were performed in the proliferative phase and confirmed by histologic criteria and transvaginal ultrasound guidance (38). The specimens were free of any endometrial pathology. During the clinical examination, ultrasound evaluation, and surgery, the presence of endometriosis or leiomyoma were excluded. Immediately after surgical removal, each sample was frozen in liquid nitrogen and kept frozen at -80 C until use to allow subsequent RNA extraction and real-time polymerase chain reaction (PCR) or paraffin embedded for immunohistochemistry.
RNA Extraction and Complementary DNA Preparation For mRNA analysis, specimens from control women (n ¼ 12), eutopic endometrium (n ¼ 8), or adenomyotic nodules (n ¼ 8) were immediately frozen and stored in liquid nitrogen. Total RNA was extracted with the SV Total RNA Isolation System (Promega) according to manufacturer's instructions. RNA was quantified by UV absorption and RNA integrity checked before downstream analysis with the FlashGel System (Lonza Group, Ltd.). For complementary DNA (cDNA) synthesis, 1 mg of total RNA was reverse transcribed using the ImProm-II Reverse Transcriptase (Promega).
Quantitative PCR Endometrial expression of target genes was analyzed by quantitative real-time PCR. The mRNA levels were measured in triplicate using the 2 Sybr Select Master Mix for CFX (Applied Biosystem) and normalized to GAPDH according to manufacturer's protocol, on a CFX Connect 96 (Bio-Rad Laboratories). Gene-specific primer sets (Supplemental Table 1, available online) were selected with Primer3-Blast or according to the published sequences (39). The PCR product identity was demonstrated by sequencing. All the primer pairs span exon–intron junctions and are located in different exons. In selected experiments, PCR products were run on 3% agarose gel to confirm the product size. For each RNA specimen, a negative control was prepared by omitting the reverse transcriptase. VOL. - NO. - / - 2015
Amplifications were carried out at 95 C for 5 minutes, followed by 10 seconds at 95 C and 30 seconds at 60 C for 40 cycles, with fluorescence detection at the end of each extension step. For each run, melting curve analysis was used to confirm the specificity of the amplified products and the absence of primer-dimer formation. Final results were expressed as fold differences in gene expression relative to the normalized calibrator, calculated by the DDCt method as follows: n-fold ¼ 2^-(DCt sampleDCt calibrator), where DCt values of the sample and calibrator were determined by subtracting the average threshold cycle (Ct) value of the transcript under investigation from the average Ct value of the GAPDH gene for each sample.
Immunofluorescent Staining For immunohistochemistry analysis, specimens from control endometrium (n ¼ 12), eutopic endometrium (n ¼ 8) or adenomyotic nodule (n ¼ 8) were used. Full-thickness sagittal sections of endometrium and adenomyotic nodule in the deep myometrium (1 1 cm) were collected. Sections (5 mm) from the formalin-fixed paraffin-embedded specimens were deparaffinized and rehydrated in a gradient ethanol series, and washed in Tris-buffered saline (20 mmol/L Tris-HCl, 150 mmol/L NaCl at pH 7.6). Tris-buffered saline was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was performed by boiling the slides in sodium citrate buffer (10 mmol/L, pH 6.0) for 15 minutes. Slides were then incubated with 10% normal goat serum diluted in 5% fetal bovine serum in a humidified chamber for 30 minutes at room temperature. After removing excess serum, primary rabbit polyclonal anti-human myostatin (Proteintech Group) or mouse monoclonal anti-human follistain (Proteintech Group) antibody (both in 1:100 dilution) was added for overnight incubation at 4 C. Normal rabbit or mouse IgG isotype was used as a negative control at the same concentrations. Then, secondary chicken anti-rabbit fluorescein isothiocyanate conjugate (FITC)-conjugated IgG (Santa Cruz Biotechnology) or chicken anti-mouse rhodamine-conjugated IgG (Santa Cruz Biotechnology) (both in dilution of 1:50) was added. Nuclei were stained with 6-diamino-2-phenylindole (DAPI) in mounting medium (Santa Cruz Biotechnology). The slides were examined using a fluorescent Olympus/IX71 microscope equipped with software OLYMPUS DP2-BSW. The intensity of the immunofluorescence was assessed with the computergenerated HSCORE ranging between 0 and 100 in 10 separate areas in each specimen using a Leica QWin image analysis system, version standard V 2.5 (Leica Imaging Systems).
Statistical Analysis Normality of the data was examined by the Shapiro-Wilk test using SigmaPlot 12.0 software (Systat Software Inc.). Data from three groups were analyzed with one-way analysis of variance (ANOVA) followed by the Holm-Sidak's post hoc test for multiple comparisons. Student's t test was performed to compare the two groups. Data were expressed as mean SD. A P value < .05 was considered significant. 3
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
FIGURE 1
Growth factors expression in adenomyosis. The messenger RNA (mRNA) expression of activin A, activin B, inhibin-a, follistatin, crypto, and myostatin in adenomyotic nodules (adenomyosis, n ¼ 8), in endometrium of the same patients with adenomyosis (eutopic, n ¼ 8), and in control endometrium (control, n ¼ 12). Fold change (y axis) represents messenger RNA expression normalized to GAPDH. *P
Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis Patrizia Carrarelli, Ph.D.,a Chih-Fen Yen, M.D.,b,c Felice Arcuri, Ph.D.,a Lucia Funghi, Ph.D.,a Claudia Tosti, M.D.,a Tzu-Hao Wang, M.D.,b,d Joseph S. Huang, M.D., Ph.D.,e and Felice Petraglia, M.D.a a Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; b Department of Obstetrics and Gynaecology, Chang Gung Memorial Hospital at Linkou and Chang Gung University College of Medicine, Taoyuan, Taiwan; c Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Taoyuan, Taiwan; d Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan; and e Department of Obstetrics and Gynaecology, The Ohio State University College of Medicine, Columbus, Ohio
Objective: To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. Design: Prospective study. Setting: University hospital. Patient(s): Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. Intervention(s): Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. Main Outcome Measure(s): Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-a, cripto), and receptors (ActRIIa, ActRIIb) were performed. Result(s): Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-a, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. Conclusion(s): The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility. (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive Use your smartphone Medicine.) to scan this QR code Key Words: Adenomyosis, nodular adenomyosis, eutopic endometrium, activin, inhibin, and connect to the myostatin, follistatin, activin receptors, crypto, infertility Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/carrarellip-activin-growth-factors-adenomyosis/
A
denomyosis is a benign uterine disorder, clinically characterized by abnormal uterine
bleeding, menorrhagia, dysmenorrheal, and infertility. Its main pathologic feature is the presence of endometrial
Received March 10, 2015; revised May 13, 2015; accepted May 24, 2015. P.C. has nothing to disclose. C.-F.Y. has nothing to disclose. F.A. has nothing to disclose. L.F. has nothing to disclose. C.T. has nothing to disclose. T.-H.W. has nothing to disclose. J.S.H. has nothing to disclose. F.P. has nothing to disclose. Patrizia Carrarelli and Chih-Fen Yen have a similar in author order. Supported by Chang Gung Memorial Hospital research grants CMRPG3C0672 to C.-F.Y. Reprint requests: Felice Petraglia, M.D., Obstetrics and Gynaecology, Department of Molecular and Developmental Medicine, University of Siena, Policlinico ‘‘Santa Maria alle Scotte,’’ Viale Bracci, 53100 Siena, Italy (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.032 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
glands and stroma diffuse within myometrium, or circumscribed in nodular aggregates of endometrial glands and stroma surrounded by myometrial hyperplasia and hypertrophy (adenomyoma) (1, 2). Clinically, endometriosis and uterine leiomyoma share similar clinical symptoms and some diagnostic features with adenomyosis (3). Adenomyosis is known to be a hormone-related disorder, characterized by high E2 receptor and impaired 1
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY P receptor expression (4, 5). Estrogens (Es) are essential for the development of adenomyosis, as they enhance the growth of endometrial glands and stroma into myometrium (1) as well as myometrial growth (6). In addition, E-associated epithelial-mesenchymal transition plays a potential role in the invasive features of endometrial cells in adenomyosis (7), with local E production induced by increased aromatase and E sulfatase activities in adenomyoma (1, 8). The epithelial-mesenchymal transition is a process that allows epithelial cells to detach from neighbor cells, to transverse the dissolving basement membranes, and to move to the extracellular matrix to reach other tissues. This process takes place during development, but it is also the mechanism used by cancer cells to colonize contiguous tissues (9). It is one of the most widely accepted proposed mechanisms underlying the myometrial invasion of the endometrium in adenomyosis. In an adenomyosis model, tissue growth, invasion, and angiogenesis occur through an annexin A2-induced E-dependent epithelial-mesenchymal transition (10). It has been proposed that, in specific cases, adenomyosis may originate from the invagination of endometrial cells into a weakened myometrium after a surgical lesion or as consequence of physiologic conditions (such as increased intrauterine pressure during pregnancy) (1, 11) or by myometrial dysfunction (1, 12). Adenomyosis is also considered an endometriosis-like disease, as endometrial invasion of myometrium underlies some of the pathogenic mechanisms of peritoneal endometriosis (i.e., cell proliferation, invasion, and neovascularization), associated with inflammation and tissue remodeling (13). Like endometriosis, adenomyosis is associated with the presence of a more invasive endometrium (14, 15). Therefore, the myometrial components may have a role in the etiology of adenomyosis as invasion is facilitated by the loss of cohesion of myometrial bundles influenced by matrix metalloproteinases (MMPs) (14, 16). Regarding molecular aspects of the pathogenesis of adenomyosis, previous studies from our and other groups have shown a deregulation of genes involved in inflammation, protease activation, and autophagy. In particular, molecules involved in extracellular matrix remodeling (MMP-2 and MMP-9) and angiogenesis, like vascular endothelial growth factor and microvessel density, are hyperexpressed in adenomyosis (17, 18). In addition, eutopic and ectopic endometria of adenomyosis showed increased interleukin expression, leading to impaired fertility in these patients (19). The transforming growth factor beta (TGF-b) superfamily includes activins, bone morphogenetic proteins, growth differentiation factors, and myostatin. Activin A (bA-subunits dimer) mediates a wide range of biological activities including wound healing, cell proliferation and differentiation, immune response, and angiogenesis (20, 21). In target cells, the binding of this growth factor to its type II receptor (ActRIIa or ActRIIb) leads to the recruitment, phosphorylation, and activation of specific type I receptor, also known as activin receptor-like kinase. Intracellular signaling is subtly regulated by cell-specific and context-dependent inactivating mechanisms by several membrane (cripto) and extracellular 2
receptor (inhibins) antagonists, and the activin-binding protein follistatin (22). In humans endometrium activin A plays a role in the menstrual cycle, with higher expression during the late secretory phase, and decidualization (23, 24). Furthermore, activin A messenger RNA (mRNA) and protein expression are significantly increased in endometrial adenocarcinoma (25), as well as in the serum and uterine washing fluid of patients affected by endometrial cancer (26). Activin A modulates the expression and secretion of interleukin-8 and vascular endothelial growth factor in cultured human endometrial stromal cells, and this mechanism is disrupted in eutopic endometrial cells from women with endometriosis (27). In addition, activin A increases endometrial cells invasiveness in an in vitro model of peritoneum, suggesting its possible role in the pathogenesis of endometriosis (28). Myostatin is another member of the TGF-b superfamily, and is a negative regulator of muscle size (29). Enlargement of the muscle mass is observed when myostatin signaling is impaired in transgenic mice carrying a dominant negative form of the myostatin receptor or in transgenic mice overexpressing follistatin, which binds and inhibits myostatin (29). Myostatin and activin A regulate myometrial cell proliferation (30, 31) and their expression is increased in uterine leiomyoma (32). Furthermore, activin A and myostatin significantly affect fibronectin, collagen1A1, and versican in primary myometrial cells and leiomyoma cells (33). Follistatin is a binding protein that stimulates muscle growth by direct inhibition of myostatin (29) and is involved in the inflammatory response by inhibiting activins and bone morphogenetic protein signaling, and is up-regulated during endothelial cell proliferation and migration (34). Follistatin expression is aberrant in endometrioma (35) and in eutopic endometrium of women with endometriosis (36), suggesting that dysfunction of this pathway may contribute to the infertility of patients with endometriosis. To our knowledge, no studies have investigated follistatin expression in adenomyosis. The present study aimed to investigate the mRNA expression of activin-related growth factors in adenomyotic tissues (nodules) and in eutopic endometrium compared with endometrium of control women. There is a possible role of these growth factors in the pathogenesis of adenomyosis.
MATERIALS AND METHODS Tissue Collection Endometrium and adenomyotic tissues (nodules) were obtained from symptomatic women, who were not pregnant and undergoing hysterectomy (age range, 38–42 years). The Institutional Review boards of Chang Gung Memorial Hospital and of the University of Siena approved this study. A written informed consent was obtained from each woman. The patient's medical history was recorded and a complete physical examination was performed based on reports of symptoms of dysmenorrhea, menorrhagia, abnormal bleeding, and pelvic pain. VOL. - NO. - / - 2015
Fertility and Sterility® The study population consisted of two groups of women: [1] patients with nodular adenomyosis (adenomyoma) (n ¼ 8) undergoing hysterectomy; and [2] control women (n ¼ 12) undergoing hysterectomy for prolapse. The diagnosis of nodular adenomyosis was carried out by two-dimensional and power Doppler pelvic and vaginal ultrasound (37). Exclusion criteria were patients receiving hormonal treatment at least 1 month before surgery, pregnancy, menopausal status, and reproductive tract cancer. Patients were excluded if preoperative ultrasound examination revealed submucous or intramural myomas (>3 or >4 cm). Adenomyotic nodules (diameter, 8–20 mm) were dissected immediately after hysterectomy, followed by histologic confirmation by a pathologist. Specimens of eutopic endometrium and from control women were collected by hysteroscopy. All surgeries were performed in the proliferative phase and confirmed by histologic criteria and transvaginal ultrasound guidance (38). The specimens were free of any endometrial pathology. During the clinical examination, ultrasound evaluation, and surgery, the presence of endometriosis or leiomyoma were excluded. Immediately after surgical removal, each sample was frozen in liquid nitrogen and kept frozen at -80 C until use to allow subsequent RNA extraction and real-time polymerase chain reaction (PCR) or paraffin embedded for immunohistochemistry.
RNA Extraction and Complementary DNA Preparation For mRNA analysis, specimens from control women (n ¼ 12), eutopic endometrium (n ¼ 8), or adenomyotic nodules (n ¼ 8) were immediately frozen and stored in liquid nitrogen. Total RNA was extracted with the SV Total RNA Isolation System (Promega) according to manufacturer's instructions. RNA was quantified by UV absorption and RNA integrity checked before downstream analysis with the FlashGel System (Lonza Group, Ltd.). For complementary DNA (cDNA) synthesis, 1 mg of total RNA was reverse transcribed using the ImProm-II Reverse Transcriptase (Promega).
Quantitative PCR Endometrial expression of target genes was analyzed by quantitative real-time PCR. The mRNA levels were measured in triplicate using the 2 Sybr Select Master Mix for CFX (Applied Biosystem) and normalized to GAPDH according to manufacturer's protocol, on a CFX Connect 96 (Bio-Rad Laboratories). Gene-specific primer sets (Supplemental Table 1, available online) were selected with Primer3-Blast or according to the published sequences (39). The PCR product identity was demonstrated by sequencing. All the primer pairs span exon–intron junctions and are located in different exons. In selected experiments, PCR products were run on 3% agarose gel to confirm the product size. For each RNA specimen, a negative control was prepared by omitting the reverse transcriptase. VOL. - NO. - / - 2015
Amplifications were carried out at 95 C for 5 minutes, followed by 10 seconds at 95 C and 30 seconds at 60 C for 40 cycles, with fluorescence detection at the end of each extension step. For each run, melting curve analysis was used to confirm the specificity of the amplified products and the absence of primer-dimer formation. Final results were expressed as fold differences in gene expression relative to the normalized calibrator, calculated by the DDCt method as follows: n-fold ¼ 2^-(DCt sampleDCt calibrator), where DCt values of the sample and calibrator were determined by subtracting the average threshold cycle (Ct) value of the transcript under investigation from the average Ct value of the GAPDH gene for each sample.
Immunofluorescent Staining For immunohistochemistry analysis, specimens from control endometrium (n ¼ 12), eutopic endometrium (n ¼ 8) or adenomyotic nodule (n ¼ 8) were used. Full-thickness sagittal sections of endometrium and adenomyotic nodule in the deep myometrium (1 1 cm) were collected. Sections (5 mm) from the formalin-fixed paraffin-embedded specimens were deparaffinized and rehydrated in a gradient ethanol series, and washed in Tris-buffered saline (20 mmol/L Tris-HCl, 150 mmol/L NaCl at pH 7.6). Tris-buffered saline was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was performed by boiling the slides in sodium citrate buffer (10 mmol/L, pH 6.0) for 15 minutes. Slides were then incubated with 10% normal goat serum diluted in 5% fetal bovine serum in a humidified chamber for 30 minutes at room temperature. After removing excess serum, primary rabbit polyclonal anti-human myostatin (Proteintech Group) or mouse monoclonal anti-human follistain (Proteintech Group) antibody (both in 1:100 dilution) was added for overnight incubation at 4 C. Normal rabbit or mouse IgG isotype was used as a negative control at the same concentrations. Then, secondary chicken anti-rabbit fluorescein isothiocyanate conjugate (FITC)-conjugated IgG (Santa Cruz Biotechnology) or chicken anti-mouse rhodamine-conjugated IgG (Santa Cruz Biotechnology) (both in dilution of 1:50) was added. Nuclei were stained with 6-diamino-2-phenylindole (DAPI) in mounting medium (Santa Cruz Biotechnology). The slides were examined using a fluorescent Olympus/IX71 microscope equipped with software OLYMPUS DP2-BSW. The intensity of the immunofluorescence was assessed with the computergenerated HSCORE ranging between 0 and 100 in 10 separate areas in each specimen using a Leica QWin image analysis system, version standard V 2.5 (Leica Imaging Systems).
Statistical Analysis Normality of the data was examined by the Shapiro-Wilk test using SigmaPlot 12.0 software (Systat Software Inc.). Data from three groups were analyzed with one-way analysis of variance (ANOVA) followed by the Holm-Sidak's post hoc test for multiple comparisons. Student's t test was performed to compare the two groups. Data were expressed as mean SD. A P value < .05 was considered significant. 3
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
FIGURE 1
Growth factors expression in adenomyosis. The messenger RNA (mRNA) expression of activin A, activin B, inhibin-a, follistatin, crypto, and myostatin in adenomyotic nodules (adenomyosis, n ¼ 8), in endometrium of the same patients with adenomyosis (eutopic, n ¼ 8), and in control endometrium (control, n ¼ 12). Fold change (y axis) represents messenger RNA expression normalized to GAPDH. *P
