Seminal Plasma Promotes Lesion Development in a Xenograft Model of Endometriosis
2015; Elsevier BV; Volume: 185; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2015.01.010
ISSN1525-2191
AutoresJonathan T. McGuane, Katherine M. Watson, Jamie Zhang, M. Zahied Johan, Zhao Wang, Gabriel Kuo, David J. Sharkey, Sarah A. Robertson, M. Louise Hull,
Tópico(s)Uterine Myomas and Treatments
ResumoThe factors that predispose one-tenth of reproductive-aged women to endometriosis are poorly understood. We determined that genetic deficiency in transforming growth factor β1 impairs endometriosis-like lesion growth in mice. Given that seminal plasma is an abundant source of transforming growth factor β, we evaluated the effect of exposure to seminal plasma on the growth of endometrial lesions. Human endometrial explants were exposed to seminal plasma or to control medium before transfer to Prkdcscid-mutant (severe combined immunodeficient) mice. Xenografts exposed to seminal plasma showed an eightfold increase in volume and a 4.3-fold increase in weight after 14 days. These increases were associated with increased proliferation of endometrial epithelial cells and enhanced survival and proliferation of human stromal cells compared with those in control lesions, in which human stromal cell persistence was negligible. Although the distribution of macrophages was altered, their number and activation status did not change in response to seminal plasma. Seminal plasma stimulated the production of a variety of cytokines in endometrial tissue, including growth-regulated oncogene, granulocyte macrophage colony-stimulating factor, and IL-1β. These data suggest that seminal plasma enhances the formation of endometriosis-like lesion via a direct effect on endometrial cell survival and proliferation, rather than via macrophage-mediated mechanisms. These findings raise the possibility that endometrial exposure to seminal plasma could contribute to endometriotic disease progression in women. The factors that predispose one-tenth of reproductive-aged women to endometriosis are poorly understood. We determined that genetic deficiency in transforming growth factor β1 impairs endometriosis-like lesion growth in mice. Given that seminal plasma is an abundant source of transforming growth factor β, we evaluated the effect of exposure to seminal plasma on the growth of endometrial lesions. Human endometrial explants were exposed to seminal plasma or to control medium before transfer to Prkdcscid-mutant (severe combined immunodeficient) mice. Xenografts exposed to seminal plasma showed an eightfold increase in volume and a 4.3-fold increase in weight after 14 days. These increases were associated with increased proliferation of endometrial epithelial cells and enhanced survival and proliferation of human stromal cells compared with those in control lesions, in which human stromal cell persistence was negligible. Although the distribution of macrophages was altered, their number and activation status did not change in response to seminal plasma. Seminal plasma stimulated the production of a variety of cytokines in endometrial tissue, including growth-regulated oncogene, granulocyte macrophage colony-stimulating factor, and IL-1β. These data suggest that seminal plasma enhances the formation of endometriosis-like lesion via a direct effect on endometrial cell survival and proliferation, rather than via macrophage-mediated mechanisms. These findings raise the possibility that endometrial exposure to seminal plasma could contribute to endometriotic disease progression in women. Endometriosis afflicts 10% of reproductive-aged women and is defined by the presence of extrauterine endometrial tissue, usually in the pelvic cavity.1Vinatier D. Orazi G. Cosson M. Dufour P. Theories of endometriosis.Eur J Obstet Gynecol Reprod Biol. 2001; 96: 21-34Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Current pharmacological therapies suppress endometriotic lesions and can ameliorate the symptoms of dysmenorrhea and chronic pelvic pain but are associated with significant side effects. Furthermore, the surgical excision of endometriotic lesions is associated with high recurrence rates of 40% to 50% at 5 years postsurgery.2Guo S.W. Recurrence of endometriosis and its control.Hum Reprod Update. 2009; 15: 441-461Crossref PubMed Scopus (445) Google Scholar Women with endometriosis are also more likely to have difficulty conceiving, suffer depression, and be at increased risk for developing cancer3Somigliana E. Vigano P. Parazzini F. Stoppelli S. Giambattista E. Vercellini P. Association between endometriosis and cancer: a comprehensive review and a critical analysis of clinical and epidemiological evidence.Gynecol Oncol. 2006; 101: 331-341Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 4Van Gorp T. Amant F. Neven P. Vergote I. Moerman P. Endometriosis and the development of malignant tumours of the pelvis. A review of literature.Best Pract Res Clin Obstet Gynaecol. 2004; 18: 349-371Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 5Nezhat F. Datta M.S. Hanson V. Pejovic T. Nezhat C. Nezhat C. The relationship of endometriosis and ovarian malignancy: a review.Fertil Steril. 2008; 90: 1559-1570Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 6Swiersz L.M. Role of endometriosis in cancer and tumor development.Ann N Y Acad Sci. 2002; 955 (discussion 293–5, 396–406): 281-292Crossref PubMed Scopus (122) Google Scholar and autoimmune disorders.7Sinaii N. Cleary S.D. Ballweg M.L. Nieman L.K. Stratton P. High rates of autoimmune and endocrine disorders, fibromyalgia, chronic fatigue syndrome and atopic diseases among women with endometriosis: a survey analysis.Hum Reprod. 2002; 17: 2715-2724Crossref PubMed Scopus (442) Google Scholar Despite the high financial and social burdens of endometriosis, the etiology and pathophysiological processes underlying the disease remain unclear.8Gao X. Outley J. Botteman M. Spalding J. Simon J.A. Pashos C.L. Economic burden of endometriosis.Fertil Steril. 2006; 86: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar Sampson's theory postulates that endometrial fragments in menstrual fluid pass in a retrograde manner through the fallopian tubes before attaching to and growing at ectopic sites.9Sampson J.A. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity.Am J Obstet Gynecol. 1927; 14: 442-446Abstract Full Text PDF Google Scholar However, this theory does not explain why only 10% of women develop endometriosis, when retrograde menstruation occurs in up to 90% of women.1Vinatier D. Orazi G. Cosson M. Dufour P. Theories of endometriosis.Eur J Obstet Gynecol Reprod Biol. 2001; 96: 21-34Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Genetic loci that confer a risk for endometriosis have been identified,10Nyholt D.R. Low S.K. Anderson C.A. Painter J.N. Uno S. Morris A.P. MacGregor S. Gordon S.D. Henders A.K. Martin N.G. Attia J. Holliday E.G. McEvoy M. Scott R.J. Kennedy S.H. Treloar S.A. Missmer S.A. Adachi S. Tanaka K. Nakamura Y. Zondervan K.T. Zembutsu H. Montgomery G.W. Genome-wide association meta-analysis identifies new endometriosis risk loci.Nat Genet. 2012; 44: 1355-1359Crossref PubMed Scopus (232) Google Scholar and various environmental, inflammatory, and immunological factors are implicated. The relative contribution of these and other causal factors remains to be established. Transforming growth factor (TGF)-β1 is a multifunctional cytokine that is elevated in endometriotic lesions, serum, and peritoneal fluid in women with endometriosis.11Pizzo A. Salmeri F.M. Ardita F.V. Sofo V. Tripepi M. Marsico S. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis.Gynecol Obstet Invest. 2002; 54: 82-87Crossref PubMed Scopus (238) Google Scholar, 12Loverro G. Maiorano E. Napoli A. Selvaggi L. Marra E. Perlino E. Transforming growth factor-beta 1 and insulin-like growth factor-1 expression in ovarian endometriotic cysts: a preliminary study.Int J Mol Med. 2001; 7: 423-429PubMed Google Scholar, 13Oosterlynck D.J. Meuleman C. Waer M. Koninckx P.R. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis.Obstet Gynecol. 1994; 83: 287-292PubMed Google Scholar When human tissue fragments were transferred to immunocompromised mice with a null mutation in Tgfb1, smaller ectopic endometriosis-like lesions developed compared with those in wild-type controls, showing that TGF-β1 bioavailability is a key determinant of lesion growth.14Hull M.L. Johan M.Z. Hodge W.L. Robertson S.A. Ingman W.V. Host-derived TGFB1 deficiency suppresses lesion development in a mouse model of endometriosis.Am J Pathol. 2012; 180: 880-887Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Seminal plasma, the acellular fraction of seminal fluid, contains several cytokines, including very high concentrations of TGF-β1, TGF-β2, and TGF-β3.15Robertson S.A. Ingman W.V. O'Leary S. Sharkey D.J. Tremellen K.P. Transforming growth factor beta—a mediator of immune deviation in seminal plasma.J Reprod Immunol. 2002; 57: 109-128Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar Seminal plasma injected i.p. increased cellular proliferation, angiogenesis, and tissue remodeling when human cervical adenocarcinoma cells were injected s.c. into nude mice.16Sutherland J.R. Sales K.J. Jabbour H.N. Katz A.A. Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo.PLoS One. 2012; 7: e33848Crossref PubMed Scopus (15) Google Scholar In vitro experiments have demonstrated increased proliferation of human endometrial epithelial cells after the addition of seminal plasma to culture media.17Gutsche S. von Wolff M. Strowitzki T. Thaler C.J. Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro.Mol Hum Reprod. 2003; 9: 785-791Crossref PubMed Scopus (104) Google Scholar, 18Khan K.N. Kitajima M. Hiraki K. Fujishita A. Sekine I. Ishimaru T. Masuzaki H. Effect of human seminal fluid on the growth of endometrial cells of women with endometriosis.Eur J Obstet Gynecol Reprod Biol. 2010; 149: 204-209Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar This finding suggests the possibility that in women, seminal plasma exposure might enhance epithelial cell proliferation in retrograde menstrual endometrium, contributing to the development or progression of endometriosis. Histological analysis indicates that endometriosis-like lesions progress from an acute inflammatory state, with neutrophil and early macrophage infiltration, to a healing and remodeling phenotype, in which macrophage and myofibroblast activity predominates.19Bromfield J.J. Schjenken J.E. Chin P.Y. Care A.S. Jasper M.J. Robertson S.A. Maternal tract factors contribute to paternal seminal fluid impact on metabolic phenotype in offspring.Proc Natl Acad Sci U S A. 2014; 111: 2200-2205Crossref PubMed Scopus (251) Google Scholar In an acute inflammatory response, phagocytic macrophages display classic (M1) activation markers such as C-C chemokine receptor (CCR)-7 and produce factors such as nitric oxide (iNOS).20Capobianco A. Rovere-Querini P. Endometriosis, a disease of the macrophage.Front Immunol. 2013; 4 (doi:10.3389/fimmu.2013.00009): 9Crossref PubMed Scopus (190) Google Scholar To prevent uncontrolled classic activation, phagocytic tags from dying cells alternatively activate macrophages via enhanced TGF-β signaling.21Gong D. Shi W. Yi S.J. Chen H. Groffen J. Heisterkamp N. TGFbeta signaling plays a critical role in promoting alternative macrophage activation.BMC Immunol. 2012; 13 (doi:10.1186/1471-2172-13-31): 31Crossref PubMed Scopus (261) Google Scholar These M2 macrophages display the alternative activation marker macrophage mannose receptor (MMR/CD206), scavenge cellular debris, and promote tissue remodeling, angiogenesis, and repair.20Capobianco A. Rovere-Querini P. Endometriosis, a disease of the macrophage.Front Immunol. 2013; 4 (doi:10.3389/fimmu.2013.00009): 9Crossref PubMed Scopus (190) Google Scholar In a mouse model, M2 macrophage activity promoted, and M1 macrophage activity suppressed, endometriosis-like lesion development.22Bacci M. Capobianco A. Monno A. Cottone L. Di Puppo F. Camisa B. Mariani M. Brignole C. Ponzoni M. Ferrari S. Panina-Bordignon P. Manfredi A.A. Rovere-Querini P. Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease.Am J Pathol. 2009; 175: 547-556Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Additionally, in TGF-β1–deficient mice, fewer macrophages were identified in ectopic endometrial lesions, suggesting that altered macrophage activity may have contributed to lesion repression. The high levels of TGF-β1 in seminal plasma could enhance lesion development by promoting an M2 macrophage healing response in ectopic endometrial tissues. In cervical and endometrial tissues exposed to seminal plasma, TGF-β1 activates the heterotetrameric TGF-βR1/TGF-βR2 receptor complex, leading to the up-regulation of several cytokines and chemokines, including granulocyte macrophage (GM) colony-stimulating factor (CSF), IL-1β, IL-6, IL-8, monocyte chemotactic protein 1, macrophage inflammatory protein 1α, and leukemia inhibitory factor.23Sharkey D.J. Macpherson A.M. Tremellen K.P. Robertson S.A. Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells.Mol Hum Reprod. 2007; 13: 491-501Crossref PubMed Scopus (212) Google Scholar, 24Sharkey D.J. Tremellen K.P. Jasper M.J. Gemzell-Danielsson K. Robertson S.A. Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus.J Immunol. 2012; 188: 2445-2454Crossref PubMed Scopus (263) Google Scholar, 25Robertson S.A. Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs.J Anim Sci. 2007; 85: E36-E44Crossref PubMed Scopus (210) Google Scholar These cytokines and chemokines recruit endometrial macrophages and dendritic cells to activate regulatory T cells and create a tolerogenic environment for an implanting embryo.19Bromfield J.J. Schjenken J.E. Chin P.Y. Care A.S. Jasper M.J. Robertson S.A. Maternal tract factors contribute to paternal seminal fluid impact on metabolic phenotype in offspring.Proc Natl Acad Sci U S A. 2014; 111: 2200-2205Crossref PubMed Scopus (251) Google Scholar Thus, it is plausible that seminal plasma–exposed endometrial tissue in retrograde menstrual fluid is more likely to escape immune surveillance, attach, and proliferate at an ectopic site in response to these chemokines. We hypothesized that exposure to seminal plasma enhances the development of endometriotic lesions via either a direct proliferative effect on endometrial cells or by increased M2 macrophage–directed healing and repair. Additionally, we hypothesized that alterations in endometrial cytokine profile induced by seminal plasma underpin these effects. A xenograft mouse model was used to test the effect of direct endometrial exposure to seminal plasma on endometriosis-like lesion growth in vivo. Immunohistochemistry (IHC) analysis was used for quantifying the effect of seminal plasma on the cellular composition and activation status of macrophages in human endometrial explants and xenografts recovered from immunocompromised mice. Finally, we explored seminal plasma–induced cytokine mediators that may influence endometriotic lesion development in human endometrial explant cultures. Approval for the use of human seminal plasma was obtained from the human ethics committees of St. Andrew's Hospital (Adelaide, SA, Australia), the University of Adelaide (Adelaide, SA, Australia), and the North Western Adelaide Health Service (Adelaide, SA, Australia) (REC 1394/03/08). The use of human endometrial tissue was approved by the human ethics committees of the Children, Youth and Women's Health Service (Adelaide, SA, Australia) (REC 2280/5/13). Animal experiments were approved by the animal ethics committees of the University of Adelaide (M-2012-080) and the Children, Youth and Women's Health Service (AE 838/4/14), and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.26National Health and Medical Research Council: Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. ed 8. National Health and Medical Research Council, Canberra2013Google Scholar Human seminal plasma was obtained from the male partners, aged 36 to 52 years (n = 6), of women undergoing treatment for female-factor infertility at Fertility SA (St. Andrew's Hospital) and healthy-proven fertile men (n = 4). Sperm analysis was conducted according to the recommendations of the World Health Organization, and samples were included only if all parameters were consistent with normal values in the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen.27World Health Organization: WHO Laboratory Manual for the examination and processing of human semen. ed 5. WHO Press, Geneva, Switzerland2010Google Scholar None of the samples displayed leukospermia or were obtained from men taking immune-deviating drugs. After liquefaction, each sample was centrifuged at 10,000 × g for 10 minutes at room temperature within 1 hour of ejaculation. The seminal plasma supernatant was then aspirated and frozen at −80°C. Equal portions of seminal plasma were subsequently thawed and pooled (to minimize the effect of variation in cytokine levels between individuals25Robertson S.A. Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs.J Anim Sci. 2007; 85: E36-E44Crossref PubMed Scopus (210) Google Scholar), aliquoted, and stored at −80°C. Our previous experience indicates that seminal plasma prepared in this way retains full bioactivity (unpublished data). Endometrial biopsy samples (n = 4) of normal histological appearance appropriate for cycle phase were collected from women aged 36 to 40 years using Pipelle suction curettage (Pipelle de Cornier, Laboratoire C.C.D., Paris, France) during diagnostic laparoscopy for infertility. Exclusion criteria included the use of hormonal contraceptives in the 3 months before surgery, or surgery for gynecological cancer. No participant had endometriosis; two were in the proliferative phase and two were in the secretory phase of the cycle. Previous work suggests cycle phase has little impact on the growth of the resulting endometriosis-like lesions (unpublished data). A separate series of endometrial biopsies was collected from five women in the secretory phase for studies of macrophage phenotype. These patients were undergoing hysteroscopy and endometrial biopsy and their endometriosis status was not determined; however, all were asymptomatic. The tissue was placed in phenol red–free Dulbecco’s modified Eagle's medium:F12 (Sigma-Aldrich, Castle Hill, NSW, Australia) with 1% each insulin-transferrin-selenium-X supplement and penicillin/streptomycin (both, Life Technologies, Mulgrave, VIC, Australia), 0.1% Ex-Cyte (Merck Millipore, Kilsyth, VIC, Australia), and 10 nmol/L 17β-estradiol (Sigma-Aldrich) as previously described.14Hull M.L. Johan M.Z. Hodge W.L. Robertson S.A. Ingman W.V. Host-derived TGFB1 deficiency suppresses lesion development in a mouse model of endometriosis.Am J Pathol. 2012; 180: 880-887Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Tissues were cut into approximately 1 mm3 fragments, placed into multiwell plates (15 fragments/well), and incubated for 24 hours in media alone or in media containing 10% seminal plasma. Female mice homozygous for the Prkdcscid mutation (severe combined immunodeficiency) were purchased from Laboratory Animal Services (University of Adelaide) at 5 weeks of age. Mice were housed under controlled light conditions (12-hour light:dark cycle) with ad libitum access to a standard diet and sterilized water. At 6 weeks of age, mice were anesthetized with 1.5% isoflurane (Veterinary Companies of Australia, Artarmon, NSW, Australia) and bilaterally ovariectomized via a single mid-dorsal incision. A 60-day release 1.5 mg 17β-estradiol pellet (Innovative Research, Sarasota, FL) was placed s.c. in the left flank via the same incision. Mepivicaine (0.02%; Ceva Animal Health, Glenorie, NSW, Australia) was administered at the incision site immediately after surgery. Buprenorphine (16 mg/mL; Reckitt Benckiser, West Ryde, NSW, Australia) was given at 0.05 mL per 10 g body weight for 12 hours postoperatively for further analgesia as needed. One to two weeks after ovariectomy, mice were anesthetized using isoflurane, and endometrial fragments were injected s.c ventrally. Mice were randomly allocated to receive human endometrial tissue that had been incubated with 10% seminal plasma for 24 hours (n = 10 mice) or control human endometrial tissue that had been incubated in media only for 24 hours (n = 11 mice). Preliminary studies of cytokine responses indicated that these conditions were optimal (data not shown), consistent with previous in vitro17Gutsche S. von Wolff M. Strowitzki T. Thaler C.J. Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro.Mol Hum Reprod. 2003; 9: 785-791Crossref PubMed Scopus (104) Google Scholar, 23Sharkey D.J. Macpherson A.M. Tremellen K.P. Robertson S.A. Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells.Mol Hum Reprod. 2007; 13: 491-501Crossref PubMed Scopus (212) Google Scholar and in vivo work.28Bruner-Tran K.L. Osteen K.G. Taylor H.S. Sokalska A. Haines K. Duleba A.J. Resveratrol inhibits development of experimental endometriosis in vivo and reduces endometrial stromal cell invasiveness in vitro.Biol Reprod. 2011; 84: 106-112Crossref PubMed Scopus (60) Google Scholar, 29Herington J.L. Crispens M.A. Carvalho-Macedo A.C. Camargos A.F. Lebovic D.I. Bruner-Tran K.L. Osteen K.G. Development and prevention of postsurgical adhesions in a chimeric mouse model of experimental endometriosis.Fertil Steril. 2011; 95: 1295-1301.e1Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The seminal plasma concentration used represents an estimate of the highest possible physiologically relevant concentration in the uterus. Each mouse received 15 tissue fragments (ie, the tissue contents of 1 seminal plasma–treated or control culture well) from one patient as previously described.30Hull M.L. Escareno C.R. Godsland J.M. Doig J.R. Johnson C.M. Phillips S.C. Smith S.K. Tavaré S. Print C.G. Charnock-Jones D.S. Endometrial-peritoneal interactions during endometriotic lesion establishment.Am J Pathol. 2008; 173: 700-715Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar The number of mice receiving transplanted tissue from a single donor varied depending on the size of the biopsy sample, but each biopsy sample was distributed approximately equally between the groups. Fourteen days post–endometrial tissue injection, mice were administered 100 μL of 10 mg/mL 5-bromo-2′-deoxyuridine (BrdU) and 5-fluoro-2′-deoxyuridine mixture (Sigma-Aldrich) i.p. and euthanized 1 hour later with 400 μL 2,2,2-tribromoethanol (Sigma-Aldrich) i.p. Endometriosis-like lesions were measured (length × width × depth) using calipers, then dissected, removed, and weighed in a blinded fashion (K.M.W.) (Figure 1A ). Tissues were transferred to 4% paraformaldehyde for 24 hours, followed by washing in phosphate-buffered saline and immersion in 70% ethanol. Tissues were dehydrated in an ascending ethanol series and transferred to xylene, then embedded in paraffin before sectioning at 5 μm. Portions of the same endometrial biopsies obtained for in vivo studies were used for analyzing cytokine production in response to seminal plasma. The additional endometrial tissues collected were prepared in triplicate for the study of macrophage phenotype in explant culture. In both experiments, an equal number of tissue fragments (1 mm3) were treated in NunclonΔ Surface multiwell plates (Thermo Scientific, Scoresby, VIC, Australia) with media containing 10% seminal plasma or media alone. Additional control wells for cytokine studies contained media with 10% seminal plasma but no tissue. Explants were incubated for 24 hours under standard cell culture conditions before conditioned media were collected for multiplex cytokine analysis and tissue was transferred to RNAlater (Sigma-Aldrich) for quantitative real-time RT-PCR or 4% paraformaldehyde for IHC analysis. Hematoxylin and eosin (Sigma-Aldrich) staining was performed on every 20th section from endometriosis-like lesions; one lesion from the control group was found not to contain glandular epithelium and was excluded from IHC analysis. The BrdU In-Situ Detection Kit (BD, North Ryde, NSW, Australia) was used to detect proliferating cells in endometriosis-like lesions as per the manufacturer's instructions. Mouse anti–human leukocyte antigen class 1 (HLA-1) (1:200 dilution; Abcam, Melbourne, VIC, Australia) was used for distinguishing human and mouse tissue in xenografts. The antibody clone used (EMR8-5) recognizes a nonpolymorphic determinant present on all HLA-1 heavy chains (ie, A, B, and C). Additional sections were stained with rat anti-mouse F4/80 IgG2aκ (1:800 dilution; eBioscience, San Diego, CA), mouse anti–α-smooth muscle actin IgG2a (1:500; Sigma-Aldrich), or rabbit anti–von Willebrand factor polyclonal antibodies (1:600; Merck Millipore, Macquarie Park, NSW, Australia) to identify macrophages (murine), myofibroblasts, and endothelial cells, respectively. Sections from both endometriosis-like lesions and endometrial explants were stained with mouse anti-human CD68 (1:50 dilution; Dako, Campbellfield, VIC, Australia), mouse anti-CD206 (1:200 dilution; R&D Systems, Minneapolis, MN), rabbit anti-CCR7 (1:60,000 dilution; Abcam), and rabbit anti-iNOS (1:1200 dilution; Santa Cruz Biotechnology, Dallas, TX) were used for identifying human macrophages and macrophages polarized to the M2 (CD206) or M1 (CCR7 and iNOS) phenotypes. Control sections were incubated with isotype-matched control antibody at the same dilution or Flex universal mouse negative control (Dako). Briefly, slides were dewaxed and rehydrated in a descending ethanol series. Antigen retrieval was performed in pH 6.0 sodium citrate buffer (10 mmol/L each citric acid and tri-sodium citrate) for 5 to 20 minutes, except for CD68 and CD206, which were performed in pH 9.0 Tris-EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA) for 5 minutes. Slides were allowed to cool for 20 minutes, washed thrice with phosphate-buffered saline, and quenched with 3% hydrogen peroxide (Sigma-Aldrich) before being incubated with 10% serum (from the species of origin of the secondary antibody) in phosphate-buffered saline with or without 1% bovine serum albumin. Sections were then incubated with the primary antibodies in phosphate-buffered saline with 2% to 10% serum overnight at 4°C. After washing, biotinylated secondary antibodies were applied for 30 to 60 minutes and detected using streptavidin-horseradish peroxidase (1:500; Dako), both at room temperature. Diaminobenzidine (Dako) was used for visualizing the stain. Slides were counterstained with hematoxylin, dehydrated, and mounted, and coverslips were applied. Morphometric analyses were performed by an assessor (J.Z. or J.T.M.) blinded to treatment group. Sections were imaged using a NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan). The proportion of BrdU-positive cells was calculated by manual counting in ImageJ software version 1.48 (NIH, Bethesda, MD) of at least 1300 cells in at least three fields per lesion. HLA-1–positive and macrophage-free stromal area was determined by measuring the glands (epithelium and lumen) and the subepithelial portion of stroma containing/devoid of substantial staining, respectively, and expressed as a percentage of the total area of each section (NanoZoomer Viewer, Hamamatsu Photonics). F4/80, von Willebrand factor, and α-smooth muscle actin staining in endometriosis-like lesions was analyzed in up to 10 fields using VideoPro 32 software version 2.1 (Leading Edge, Adelaide, SA, Australia) and expressed as percentage of positive area. CD68, CD206, CCR7, and iNOS were similarly measured in multiple fields encompassing the entire endometrial explant tissue section. A custom Milliplex MAP Human Cytokine/Chemokine 96-well multiplex assay (Merck Millipore) was designed and run on a Luminex 200 System (Luminex Corp., Austin, TX) according to the manufacturer's instructions. The minimal detectable limits of the 20 analytes ranged from 0.4 to 26.3 pg/mL. A separate assay (R&D Systems) was used for assessing IL-4 and macrophage CSF levels in conditioned media. Samples that were below the minimal detectable limits were excluded; only analytes for which >50% of samples tested were above the minimal detectable limits were considered for statistical analysis. The mean intra- and interassay coefficients of variation for each analyte were 13% and 5%, respectively. Total RNA was extracted from seminal plasma–treated and control explants using TRIzol reagent (Life Technologies) with RNeasy column purification (Qiagen, Chadstone, VIC, Australia) as per the manufacturer's instructions. Spectrophotometric analysis on a NanoDrop ND-1000 (Thermo Scientific) confirmed the purity of the resultant sample [A260:A280, 1.9 ± 0.1 (means ± SEM)]. Total RNA (60 ng) was reverse-transcribed in a volume of 20 μL using SuperScript III with 250 ng random primers (both, Life Technologies) as per the manufacturer's instructions. PCR was performed in triplicate for ACTB and in duplicate for cytokine genes using Power SYBR Green Master Mix on an Applied Biosystems 7900HT Real-Time PCR system (both, Life Technologies). Cytokine gene fold-change in response to treatment with seminal plasma was c
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