TGF-β1 in Seminal Plasma Promotes Endometrial Mesenchymal Stem Cell Growth via p42/44 and Akt Pathway in Endometriosis

Background: The cause of endometriosis, which is characterized by the existence of functional endometrial tissue outside the uterine cavity, is poorly understood. Seminal plasma (SP) is rich in multiple cytokines may promote endometrial tissue survival. Methods: We evaluated the effect of SP on growth of endometrial mesenchymal stem cells (MSCs) from women with endometriosis (E-MSCs), and women without endometriosis (NE-MSCs). Additionally, effects of SP on endometriotic implants were analyzed using a xenograft model of endometriosis in immunode ﬁ cient nude mice. Results: Proliferation, cell foci formation, cell cycle progression, and growth marker expression of E- and NE-MSCs were promoted by SP. SP signi ﬁ cantly stimulated phosphorylation of p42/44 and Akt and increased expression of the proliferation marker in E- and NE-MSCs, which was attenuated by transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542, Akt inhibitor LY294002, or p42/44 MAPK inhibitor PD98059. SP enhanced CDK2 and CDK6 expression and accelerated cell cycle progression in E-MSCs. Xenografts exposed to SP exhibited a three-fold increase in volume and four-fold increase in weight after 14 days. Conclusions: Our ndings demonstrate that TGF-β1 in SP may promote endometrial tissue survival. These effects may be mediated through activation of TGF-β1, Akt, and p42/44 signaling, which enhances CDK2 and CDK6 expression and accelerates cell cycle progression. undergo osteogenesis. Mineralized calcium was deposited after induction, by positive Alizarin Red S and alkaline phosphatase staining. Following adipogenic induction conditions, Red staining accumulation of lipids, the successful isolation of MSCs.


Background
Endometriosis, a major cause of infertility, is a prevalent gynecologic disease characterized by the existence of functional endometrial tissue outside the uterine cavity, which causes pain and affects fertility in almost 10% of reproductive women 1,2 . The precise mechanisms that cause endometriosis remain unknown. Sampson's theory posits that endometriotic implants arise from retrograde menstruation of endometrial tissue through the fallopian tubes into the peritoneal cavity 3 . The human endometrium is a unique tissue exhibiting regular cyclical periods of growth, denudation, and renewal.
Mesenchymal stem cells (MSCs) from endometrium have been characterized as multipotent cells of mesenchymal origin that can differentiate into a number of different cell types, and were implicated in cyclic endometrial regeneration 4 . Endometrial MSCs have been detected in the basalis layer of the endometrium and endometriotic implants 5 , and may be responsible for the development of endometriosis 5 . However, factors capable of triggering the proliferation of endometrial MSCs require further study.
Endometriosis is relatively common in women of childbearing age. Sexual activity in females allows contact with semen, which contains sperm and seminal plasma (SP) rich in numerous cytokines. SP can induce epithelial-mesenchymal transdifferentiation and the expression of myo broblastic metaplasia markers in endometriotic and endometrial cells 6 . In the absence of a barrier contraceptive, the growth of endometrium can be further modulated by mediators present in SP, which is derived from sexual activity.
Previous research showed that SP can promote endometriotic development 7,8 and induce global transcriptomic changes that increase the risk for endometriosis 9 . Moreover, patients with endometriosis may be oversensitive to stimulation from SP. In vitro experiments showed that endometrial epithelial and stromal cells had an enhanced proliferative response to SP compared with cells from women without endometriosis 7 . In vivo experiments showed that SP enhanced endometrial survival and facilitated endometriotic lesion development 8 . Although a few studies have focused on the role of SP in promoting endometriotic development, downstream signaling mechanisms are not fully understood. SP may also activate the development of endometrial MSCs, which are sensitive to extracellular environments that promote endometriotic lesion survival.
As the acellular fraction of seminal uid, SP is rich in cytokines, chemokines, growth-factors, and steroid hormones. In particular, SP contains high concentrations of TGF-β1, TGF-β2, and TGF-β3 10 . TGF-β1 is a multifunctional regulatory cytokine involved in several cellular functions, including proliferation, migration, invasion, and differentiation 11 . TGF-β1 expression is increased in peritoneal uid and lesions of women with endometriosis 12 . Ibrahim et al. described novel evidence that even when semen is washed twice, SP is still detectable 6 . After sexual intercourse, SP components were found to enter into the endometrial bed or peritoneal bed as a result of hematogenous dissemination or direct tissue perfusion through the anterior or posterior vaginal fornix 13 . Therefore, it is possible that TGF-β1 in SP, which comes into contact with the endometrial bed after unprotected sexual intercourse, can induce biological changes in those tissues and cells. Here, we explored whether SP could activate MSCs in endometrium through TGF-β1.
Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways play important roles in stimulating proliferation of endometriotic cells, production of in ammatory factors, and neovascularization. Honda et al. reported activation of PI3K/Akt and MAPK signaling pathways in endometriosis 14 . MAPKs are activated by multiple extracellular signals that promote cell proliferation 15 , whereas Akt phosphorylation is mediated by PI3K in response to numerous extracellular signals 16 . Both MAPK and PI3K/Akt pathways are crucial for the regulation of cell survival and proliferation 15,16 . The molecular mechanisms of TGF-β1-regulated MSC proliferation have been extensively studied. Here, we determined whether MAPK and Akt pathway activation is involved in the stimulation of endometrial MSC proliferation. In addition, the TGF-β receptor inhibitor SB431542, Akt inhibitor LY294002, and p42/44 MAPK inhibitor PD98058 were examined for their ability to reverse the effect of SP on endometrial MSC proliferation.
Understanding whether SP can activate the proliferation of MSCs is of great importance because unprotected sexual intercourse is common. In the present study, we found that TGF-β1 in SP promotes the growth of endometrial MSCs via p42/44 and Akt pathways, indicating a potential prevention and treatment strategy for endometriosis.

Patients
This study was approved by the ethics committee of Sir Run Run Shaw Hospital (Hangzhou, China). The patient group consisted of women with endometriosis (stage III-IV) undergoing laparoscopy for pain or infertility (n = 6). The control group consisted of women with no endometriosis undergoing laparoscopy for tubal disease (n = 6). Human SP was obtained from male partners [(n = 15), aged 25 to 39 years] of women undergoing treatment for female-factor infertility at Sir Run Run Shaw Hospital in China and healthy fertile men. None of the donors had received hormonal therapy for at least 6 months prior to surgery. Participants had regular menstrual cycles (27-32 days

Isolation of MSCs
To isolate and generate MSC lines from the endometrium of women with endometriosis (E-MSCs) and without endometriosis (NE-MSCs), endometrial tissue biopsies were obtained just prior to surgery using an endometrial suction catheter (Lilycleaner, Ningbo, China). Cells were isolated from biopsy specimens of the endometrium and seeded in triplicate at low density (approximately 200 cells per 100-mm dish) in Dulbecco's Modi ed Eagle's Medium with F12 Supplement (DMEM/F12; Gino167 Biological, Hangzhou, China). Large colonies were isolated and trypsinized into single-cell suspensions following incubation for 21 days. The resulting cell suspensions were diluted and seeded into 96-well plates at a density of approximately one cell per well. After 14 days in culture, clonally derived proliferating colonies were trypsinized and individually cultured in individual 100-mm dishes. Cells were allowed to grow in DMEM/F12 with 10% fetal bovine serum (FBS), and adherent cells were cultured until they reached 80%-90% con uence. Cells were trypsinized, subcultured, and used for experiments during passages two to four.

Multipotent differentiation
The multipotency of endometrial MSCs for osteogenesis and adipogenesis was determined as previously described 18 . Brie y, to induce osteogenesis, MSCs were plated at a density of 5 ×

SP Collection
Semen samples obtained from 15 donors were allowed to liquefy for 2 hours. After liquefaction, each sample was centrifuged at 700 × g for 10 minutes at room temperature within 2 hours of ejaculation. The supernatant was centrifuged at 10,000 × g for 30 minutes to remove spermatozoa. The collected samples were pooled, ltered and concentrated using Centricon Plus-20 centrifugal concentration tubes (3,000 NMWL; Millipore, Cork, Ireland) ( Supplementary Fig. S1A). Acid activation of cytokines in SP was achieved by treating concentrated SP with 1 N HCl, followed by incubation at room temperature for 10 minutes. SP was then neutralized with 1.2 N NaOH/0.5 M HEPES before resuspending in DMEM/F12. The treated SP was aspirated and frozen at -80 °C. Prior to use, concentrated SP was thawed on ice, resuspended, and diluted 1:100 with DMEM/F12 for in vitro studies, or 1:500 with sterile-ltered phosphate-buffered saline (PBS) for in vivo studies.

Treatment of cells
Cells were seeded into 96-well plates at a density of 2 × 10 3 cells per well for proliferation analyses, and 5 × 10 4 cells per 60-mm dish for western blotting (WB). For cell proliferation analyses, cells were cultured at 37 °C for 1 day in DMEM/F12 containing 10% FBS, followed by incubation with control (DMEM/F12 + 2% FBS) or SP containing 2% FBS for the indicated time. For foci formation assay, ow cytometry analysis, and WB, cells were cultured at 37 °C for 1 day and then incubated for 1 day in serum-free culture media.
Subsequently, culture media were replaced and cells were treated with vehicle control or SP. Foci formation assay was performed after 2 days of culture. WB analysis was performed after 2 days of culture or at speci ed time (0, 1, 2 and 6 hours).
Cell-cycle parameters were determined by ow cytometry of propidium iodide (PI)-stained endometrial MSCs 19 . Brie y, MSCs (2 × 10 6 ) were xed in 70% ethanol at 4 °C overnight. The ethanol was discarded and MSCs were resuspended in PBS. Cell pellets were stained with 50 µg/ml PI and 100 μg/ml RNase A in PBS for 30 minutes at room temperature. After washing with PBS, MSCs were analyzed by an Epics XL ow cytometer.
Flow cytometric analysis of apoptosis markers was performed. Endometrial MSCs were washed in cold PBS and incubated with FITC-conjugated Annexin and PI solution (Invitrogen, Carlsbad, CA, USA) at room temperature for 15 minutes in the dark. Stained cells were analyzed using an Epics XL ow cytometer.
Cell proliferation assay Cell proliferation was determined using a Cell Counting Kit 8 (CCK-8) assay. Endometrial MSCs were seeded into 96-well plates at a density of 2 × 10 3 cells per well. After cell attachment, control (phenol redfree DMEM/F12 containing 2% FBS) or resuspended SP containing 2% FBS was added to cells. To evaluate changes in cell proliferation, cell counting was performed using a CCK-8 kit (Tojindo, Shanghai, China) according to the manufacturer's instructions.

Colony-forming assay
MSCs were seeded into six-well culture plates at a density of 1 × 10 2 cells per well and incubated at 37 °C for 14 days. Subsequently, MSCs were stained with a crystal violet solution and imaged with a microscope equipped with a digital camera.

Mouse model of endometriosis
Mature female athymic nude mice (6 weeks old) purchased from Shanghai Laboratory Animal Co. Ltd. (Shanghai, China) were housed in a pathogen-free and climate-controlled environment (23-25 °C) with regulated 12-hour light/dark cycles. One week was allowed for acclimatization prior to experimental proceedings. Three days prior to endometrial transplantation, mice received a daily intraperitoneal injection of 200 µg/kg 17β-estradiol. The nude mouse model of endometriosis was established as previously described 18,20 . Proliferative-phase human endometrial fragments (1-2 mm 3 ) from endometriosis patients were washed with sterile serum-free DMEM/F-12 culture medium. Human endometrial fragments were implanted subcutaneously into mice under general anesthesia. To achieve higher local concentrations, subcutaneous injection was employed as the administration route based on previous animal experiments 18,20,21 (Supplementary Fig. S1B). Subcutaneous injections of TGF-β1 (40 ng/g), SP, or the TGF-β receptor inhibitor SB431542 (10 ug/g) were employed. Daily intralesional injections were started on the rst day after endometrial tissue implantation and continued for 14 days.
Mice were then randomly divided into four groups: control, TGF-β1, SP, and SP+SB431542. Endometrial tissues were harvested and measured after 14 days of intralesion injections commencing from day 1 post-implantation. Treatment dosages used were determined by preliminary experiments. Endometriotic implants were collected, xed in 10% formalin-acetic acid, and embedded in para n for histopathological examination. Immunohistochemical staining was performed according to well-established protocols 1 . Anti-human leukocyte antigen (HLA) antibody (1:100; Abcam) was used to identify human cells within xenograft tissue. Tissue sections were also incubated with a polyclonal antibody for the proliferative marker Ki67 (1:100; Abcam). Staining scores and parameters were calculated using a computerized image analysis system, as previously described 18 . Areas of positive staining were calculated. In addition, the whole area of the endometriotic implant in each section was analyzed.
Data analysis and statistics SPSS version 16.0 (SPSS, Chicago, IL) was used to analyze complete datasets. Student's t test, one-way analysis of variance, or Scheffe's general linear model of repeated measures method was used to compare differences between treatment groups. Statistical signi cance was de ned as P < 0.05.

Isolation and characterization of NE-and E-MSCs
Isolated NE-MSCs and E-MSCs were kept in culture until passage 20, indicating a self-renewal ability typically associated with MSCs 22 . To verify NE-and E-MSC phenotypes, we evaluated the expression of positive mesenchymal markers CD44, CD90, CD73, and CD29, as well as negative MSC markers CD117, CD34, CD45, and HLA-DR. Mesenchymal markers were highly expressed in more than 90% of NE-and E-MSCs, while mesenchymal-negative markers were observed in less than 10% of NE-and E-MSCs ( Supplementary Fig. S2A). An osteogenic differentiation assay con rmed that the majority of NE-and E-MSCs had the capacity to undergo osteogenesis. Mineralized calcium was deposited after osteogenic induction, as con rmed by positive Alizarin Red S and alkaline phosphatase staining. Following adipogenic induction conditions, Oil Red O staining demonstrated the accumulation of lipids, indicating MSCs had differentiated into adipogenic lineages (Supplementary Fig. S2B). Collectively, these results indicated the successful isolation of MSCs.

SP promoted NE-and E-MSC proliferation, cell foci formation, cell cycle progression, and growth marker expression
To investigate the effect of SP on MSC proliferation, NE-and E-MSCs were cultured with SP in vitro, and CCK-8 assays were performed. SP treatment signi cantly increased the proliferation of MSCs compared with controls (Fig. 1A). In addition, SP could signi cantly increase the ability of MSCs to form colonies, as demonstrated by the foci formation assay (Fig. 1B). Flow cytometry was used to determine the in uence of SP on the cell cycle progression of NE-and E-MSCs. Compared with the control group, the number of cells in G0/G1 of the SP-treated group was decreased, and the number of S-phase cells was elevated by SP treatment; however, the distribution of G2/M phase cells did not change signi cantly (Fig. 1C). SP promoted MSC entrance into S phase from G0/G1, which was considered indicative of cell growth. Apoptosis of MSCs was determined by ow cytometric analysis of annexin V FITC/PI staining. There was no signi cant reduction or increase in the percentage of live cells in the SP-treated group compared with the control group. After treatment with SP or vehicle, a small fraction of apoptotic cells was observed.
The percentage of apoptotic cells was similar in SP-treated and control groups (Fig. 1D). We also evaluated the effect of SP on expression of proliferating cell nuclear antigen (PCNA), an essential component of DNA polymerase δ 23 , and the antiapoptotic protein Bcl-2. WB assay results demonstrated that SP elevated the protein expression of PCNA (Fig. 1C), while Bcl-2 protein expression was unchanged (Fig. 1E). These results indicate that SP activation promoted the proliferation of MSCs and accelerated their cell cycle.

TGF-β1 in SP promoted MSC proliferation via phosphorylation of p42/44 and Akt
We next examined the role of p42/44 and Akt pathways, which control cell proliferation, differentiation, and survival 24 . Expression of p-p42/44, T-p42/44, p-Akt-S, p-Akt-T, T-AKT, and β-tubulin was assessed by WB. We found that SP stimulated phosphorylation of p42/44 and Akt both at serine 473 (Ser473) and threonine 308 (Thr308) at various time points (1, 2, and 6 hours) after incubation with SP, indicating activation of p42/44 and Akt pathways ( Fig. 2A). Next, the involvement of p42/44 and/or Akt signaling in SP-enhanced proliferation of MSCs was examined. SP contains high concentrations of TGF-β1, which is involved in the activation of p42/44 and/or Akt signaling pathways 25 . TGF-β1 (2 ng/ml), SP, SP with the TGF-β receptor inhibitor SB431542, SP with the AKT inhibitor LY294002, or SP with the p42/44 MAPK inhibitor PD98059 were used to stimulate NE-and E-MSCs, and determine whether MSC-secreted TGF-β1 contributes to cell proliferation through p42/44 or Akt pathways. Pretreatment with inhibitors was conducted prior to stimulation with SP. TGF-β1, SP signi cantly increased the expression of the proliferation marker PCNA. However, addition of the TGF-β receptor inhibitor SB431542, Akt inhibitor LY294002, or p42/44 MAPK inhibitor PD98059 markedly attenuated PCNA protein expression in SPtreated MSCs (Fig. 2B). These results indicate that p42/44 and Akt signaling are involved in the enhanced MSC proliferation caused by SP activation.

SP induced modulation of cell cycle-related protein CDK2 and CDK6 expression via p42/44 and Akt signaling
The cell cycle is driven, in part, by cyclin dependent kinases (CDKs) and their activation by cyclins 26 . Cyclin D/CDK4/CDK6 activity occurs in mid to late G1 phase, upstream of CDK2/cyclin E activity. The complexes formed by cyclins and CDKs can regulate kinase activity and progression through G1/S phase. Cyclin A may be required for both S phase and M phase 27 . In the present study, CDK2 and CDK6 protein levels were higher in SP-treated E-MSCs whereas, CDK4, cyclin A2, cyclin D1, and cyclin E2 protein levels remained largely unchanged (Fig. 3A). SP activation may accelerate the cell cycle by increasing expression of CDK2 and CDK6, resulting in enhanced E-MSC proliferation. We also examined effects of the AKT inhibitor LY294002 and p42/44 MAP kinase inhibitor PD98059 on SP-induced cell cycle-related protein expression. SP treatment increased CDK2 protein levels, which, conversely, were markedly attenuated by LY294002. CDK6 protein levels were also markedly attenuated by LY294002 and PD98059 (Fig. 3B). These results indicate that SP-induced increases in p42/44 and Akt signaling may result in upregulation of CDK2 and CDK6 expression, consequently accelerating the cell cycle.

Exposure to SP promoted endometriosis-like lesion development in mice
To con rm our hypothesis, an in vivo SCID mouse model was used. All mice developed endometriosis-like lesions showing typical characteristics of endometriosis with glandular structures and stroma. Endometriosis-like lesions harvested from mice treated with TGF-β1 and SP were found to be signi cantly larger and heavier than lesions treated with vehicle alone (Fig. 4A-C). Xenografts exposed to SP showed a three-fold increase in volume and four-fold increase in weight after 14 days. Endometriosis-like lesions may contain both donor human and host mouse tissue. Part of the xenograft tissue stained positively for HLA, indicating its human origin. There was a signi cantly greater proportion of donor tissue in endometriosis-like lesions from TGF-β1 and SP groups (Fig. 4D, F). Moreover, cells positive for Ki67, a marker for proliferation 28 , were signi cantly more numerous in endometriosis-like lesions after TGF-β1 and SP treatment compared with controls (Fig. 2E, F). However, the growth-promoting effect of SP was attenuated by the TGF-β receptor inhibitor SB431542 (Fig. 4A-F). These results indicate that SP directly supports the proliferation of cells of endometrial origin via TGF-β1.

Discussion
Endometriosis is one of the most common gynecological disorders. Its pathogenesis remains unknown despite extensive study. Studying the role of stem cells in the etiology of endometriosis may illuminate some of the factors that initiate this disease. Many studies have demonstrated the presence of MSCs in endometrium and menstrual blood 16,29 . Recent studies showed that menstrual blood-derived MSCs from women with endometriosis showed a higher proliferative capacity compared with MSCs from women without endometriosis; they also revealed different phenotypic and functional characteristics 30 . Aggressive overgrowth of MSCs may be a cause of endometriosis. SP may in uence endometriotic lesion development in human endometrial explant cultures 8 . The endometrium has unique regenerative properties arising from the existence of MSCs. Once in the abdominal cavity, endometrial MSCs can proliferate, invade, and differentiate into endometrial cells, nally generating ectopic implants 31 . SP contains high concentrations of cytokines, such as TGF-β1 10 , a multifunctional regulatory cytokine. Indeed, the susceptibility of MSCs to SP may be a cause of endometriosis.
Our ndings demonstrate that SP can induce the proliferation of NE-MSCs and E-MSCs. SP-treated MSCs exhibited accelerated growth, cell foci formation, and cell cycle progression. In addition, SP treatment signi cantly increased protein levels of the proliferation marker PCNA. Thus, our results show that SP could enhance cell proliferation, which con rms the work of others 1,9 . To determine how SP enhances cell proliferation, we evaluated TGF-β1, p42/44, and Akt signaling pathways. P42/44, a part of the MAPK cascade, can be activated by extracellular or intracellular factors. Activated p42/44 is transported into the nucleus, whereby it increases proliferation-related gene expression 32 . Activation of Akt signaling is a vital regulator of cell survival 15 , while both p42/44 and Akt signaling are critical to cell proliferation. We found that phosphorylated p42/44 phosphorylates Akt both at Thr308 and Ser473, which were both highly expressed in SP-treated MSCs compared with controls, indicating activation of p42/44 and Akt signaling. To further explore the role of TGF-β1, p42/44, and Akt signaling pathways on SP-driven cell proliferation, signaling was inhibited by the TGF-β receptor inhibitor SB431542, Akt inhibitor LY294002, or p42/44 MAPK inhibitor PD98059. Inhibition of this signaling markedly attenuated SP-mediated proliferation, indicating that TGF-β1 can modulate Akt and p42/44 signaling pathways in cell growth and proliferation.
Cell cycle progression is dependent, in part, on tightly regulated activity of cell cycle-related proteins. By assessing the expression of such proteins, we demonstrated that increased protein expression of CDK2 and CDK6 was attenuated by the Akt inhibitor LY294002 and p42/44 MAPK inhibitor PD98059.
Complexes with CDK2/CDK6 and cyclins in uence progression through G1/S phase 27 . The activation of p42/44 signal can increase the expression of CDKs and accelerate the cell cycle 33 , which is in accordance with our results indicating that Akt and p42/44 signaling pathways have a cooperative effect on SP-enhanced proliferation of MSCs by increasing the expression of proteins regulating G1/S progression.
In vivo experiments suggest that treatment with the TGF-β receptor inhibitor SB431542 may retard the progression of endometriosis, which is conversely accelerated by TGF-β1 and SP. This result con rms a previous study conducted in a mouse model of adenocarcinoma tumor, whereby SP enhanced lesion proliferation and growth 1 . These ndings and previous in vitro results indicate a signi cant role for SP in the progression of endometriosis.
SP contains numerous types of in ammatory agents, such as TGF-β, prostaglandins, and glycoprotein signaling molecules including growth factors and cytokines. These molecules bind to their target cell to modulate gene expression and cell function. One limitation of our research is that we have not identi ed all of the signaling molecules in SP responsible for orchestrating the effects we observed in vivo and in vitro. However, these effects may result from the integration of all signaling in SP though TGF-β, making it potentially one of the most important factors.

Conclusion
We showed that SP promotes NE-and E-MSC proliferation, cell foci formation, cell cycle progression, and growth marker expression. In vitro experiments showed that this effect may be mediated through activation of TGF-β1, Akt and p42/44 signaling, which enhances the expression of CDK2 and CDK6, thus accelerating cell cycle progression. This study also provides in vivo evidence that TGF-β1, which is present in SP, could accelerate endometriosis. Thus, TGF-β1 in SP may promote growth of MSCs via p42/44 and Akt pathways.   SP-driven endometriosis-like lesion development in mice. The volume (A) and weight (B) of endometriosis-like lesions harvested from mice treated with vehicle, TGF-β1, SP, or SP with the TGF-β receptor inhibitor SB431542. Xenografts exposed to SP showed a three-fold increase in volume and fourfold increase in weight after 14 days. (C) Images of endometriosis-like lesions harvested from the sacri ced animal. (D) Staining scores of HLA (indicating human origin) and Ki67 (marker for proliferation) immunohistochemistry in an endometriosis-like lesion. a: P < 0.05 versus control-treated mice. c: P < 0.05 versus SP-treated mice. V: vehicle-treated mice; (n = 6). Scale bars = 100 μm.

Supplementary Files
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