{"paper_id":"69036a44-e3a3-41c8-ae17-47bb816ac437","body_text":"The Effect of Mesenchymal Stem Cells on\nFertility in Experimental Retrocervical\nEndometriosis\nO efeito das células-tronco mesenquimais na fertilidade\nem endometriose retrocervical experimental\nJaqueline Pedroso de Abreu 1 Carmen Lúcia Kuniyoshi Rebelatto 2 Carolina Antunes Savari 1\nLuiz Guilherme Achcar Capriglione 3 Lye Miyague 3 Lucia de Noronha 1 Vivian Ferreira do Amaral 4\n1 Center for Health and Biological Sciences, Pontifícia Universidade\nCatólica do Paraná (PUC -PR), Escola de Medicina, Curitiba, Paraná,\nBrazil\n2 Experimental Laboratory of Cell Culture, PUC -PR, Curitiba, Paraná,\nBrazil\n3 Center for Health and Biological Sciences, Escola de Ciências\nAgrárias e Medicina Veterinária, PUC -PR, Curitiba, Paraná, Brazil\n4 Department of Obstetrics and Gynecology, Universidade Federal do\nParaná (UFPR), Curitiba, Paraná, Brazil\nRev Bras Ginecol Obstet 2017;39:217 –223.\nAddress for correspondnece Jaqueline Pedroso de Abreu, MSc,\nDepartamento de Tocoginecologia – HCUFPR, Gen. Carneiro,\n181 - Alto da Glória, 80060-900, Curitiba, PR, Brazil\n(e-mail: jaquelinepabreu@gmail.com).\nKeywords\n► endometriosis\n► mesenchymal stem\ncell\n► fertility\n► rabbits\nAbstract Purpose To evaluate the effect of mesenchymal stem cells (MSCs) on fertility in\nexperimental retrocervical endometriosis.\nMethods A total of 27 New Zealand rabbits were divided into three groups:\nendometriosis, in which endometrial implants were created; mesenchymal, in which\nMSCs were applied in addition to the creation of endometrial implants; and control, the\ngroup without endometriosis. Fisher ’se x a c tt e s tw a sp e r f o r m e dt oc o m p a r et h e\ndichotomous qualitative variables among the groups. The quantitative variables were\ncompared by the nonparametric Mann-Whitney and Kruskal-Wallis tests. The Mann-\nWhitney test was used for post-hoc multiple comparison with Boniferroni correction.\nResults Regarding the beginning of the fertile period, the three groups had medians\nof 14 /C6 12.7, 40 /C6 5, and 33 /C6 8.9 days respectively ( p ¼ 0.005). With regard to fertility\n(number of pregnancies), the endometriosis and control groups showed a rate of\n77.78%, whereas the mesenchymal group showed a rate of 11.20% ( p ¼ 0.015). No\ndifferences in Keenan ’s histological classi fication were observed among the groups\n(p ¼ 0.730). With regard to the macroscopic appearance of the lesions, the mesen-\nchymal group showed the most pelvic adhesions.\nConclusion The use of MSCs in endometriosis negatively contributed to fertility,\nsuggesting the role of these cells in the development of this disease.\nreceived\nOctober 5, 2016\naccepted after revision\nFebruary 2, 2017\npublished online\nApril 11, 2017\nDOI https://doi.org/\n10.1055/s-0037-1601484.\nISSN 0100-7203.\nCopyright © 2017 by Thieme Revinter\nPublicações Ltda, Rio de Janeiro, Brazil\nTHIEME\nOriginal Article 217\n\n\nIntroduction\nEndometriosis is a disease characterized by the presence of\nglands and/or endometrial stroma outside the uterine cav-\nity.\n1 Endometriosis can be macroscopically divided into\nthree clinically distinct entities: super ficial peritoneal en-\ndometriosis, ovarian endometriosis (endometrioma), and\ndeep endometriosis. 2,3 Deep endometriosis is estimated to\noccur in /C24 20% of women with the disease. 4\nThe most common symptoms of endometriosis are dysme-\nnorrhea, dyspareunia, chronic pelvic pain, and infertility, 5,6\nand the deep form of the disease is often related to infertility.5,6\nOne study reported the detection of endometriosis in 20–50%\nof women who underwent investigations for infertility.7\nCurrently, groups of scientists are researching the presence\nof progenitor stem cells in the endometrium and the correlation\nof this phenomenon with endometrial regeneration and men-\nstrual cyclicity.8–11 In 2004, a research group led by Gargett 12\ndemonstrated the presence of a small group of cells exhibiting\nclonogenic capacity in the human endometrium. 11,13 In the\nsame year, another group led by Taylor proposed that bone\nmarrow cells in humans could participate in the endometrial\nregeneration process in recipient patients.\n12,14\nVidane et al 15 suggested that mesenchymal cells derived\nfrom the bone marrow also contribute to endometrial\ngrowth and play a role in the progression of endometriosis.\nEndometriosis is a disease with in flammatory characteris-\ntics.2,10 Furthermore, mesenchymal stem cells (MSCs) release\nfactors with the following trophic properties: immunomodu-\nlatory, angiogenic, and antiapoptotic.16,17 These cells appear to\nneed an inflammatory environment for exerting their immu-\nnomodulatory function,18 and such an environment exists in\nendometriosis.\nMesenchymal stem cells are considered non-immuno-\ngenic, but they can differentiate into various tissues and\nsecrete immunomodulatory and regenerative/reparative fac -\ntors. Therefore, MSCs are considered optimal for cell ther-\napy.15 These cells are primarily used for regeneration; their\neffects on the immune system and the secretion of growth\nf a c t o r sh a v eo p e n e dfields in the clinical research.\n16\nThe complexity of the pathophysiology of endometriosis,\nthe heterogeneity of the disease in humans, and the effect of\nmesenchymal cells as immunomodulators are still contro-\nversial.\n19 In this context, female rabbits are a good choice as\nexperimental models. They have homogeneous lesions —that\nis, generally solid and hemorrhagic masses —that can be\neasily created by autograft of endometrial fragments or by\nthe opening and exposure of the endometrial cavity. 20\nThe objective of this study was to evaluate the possible\ncontribution of MSCs for the development of endometriosis\nand its effects on fertility through induced lesions in rabbits.\nMethods\nThis experimental study was conducted after approval by the\nAnimal Ethics Committee of the university (CEUA- PUCPR,\nproject no. 710 C).A total of 29 adult female New Zealand\nrabbits (6 –8 months of age) weighing between 3 and 4 kg\nwere used. This total included 27 rabbits used for the sample\nand 2 for bone marrow collection.\nResumo Objetivo Avaliar o efeito das células-tronco mesenquimais sobre a fertilidade na\nendometriose retrocervical experimental.\nMétodos Um total de 27 coelhas da raça Nova Zelândia foram divididas em três\ngrupos: endometriose, em que os implantes endometriais foram criados; mesenqui-\nmal, em que as células-tronco mesenquimai s foram aplicadas complementarmente à\ncriação implantes endometriais; e controle , sem endometriose. O teste exato de Fisher\nfoi realizado para comparar variáveis dicotômicas qualitativas entre os grupos. As\nvariáveis quantitativas foram comparadas pelos testes não paramétricos de Mann-\nWhitney e Kruskal-Wallis. O teste de Mann-Whitney foi utilizado para a comparação\nmúltipla pós-hoc com correção de Boniferroni.\nResultados e mr e l a ç ã oa oi n í c i od op e r í o d of é r t i l ,o sg r u p o se n d o m e t r i o s e ,m e s e n -\nquimal e controle tiveram medianas de 14 /C6 12,7; 40 /C6 5; e 33 /C6 8,9 dias,\nrespectivamente ( p ¼ 0,005). Sobre a taxa de fertilida de (número de gravidezes), os\ngrupos endometriose e controle mostraram uma taxa de 77,78%, enquanto o grupo\nmesenquimal mostrou uma taxa de 11,20% ( p ¼ 0,015). Não foram observadas\ndiferenças na classi ficação histológica de Keenan entre os grupos ( p ¼ 0,730). No\nque diz respeito à aparência macroscópica das lesões, o grupo mesenquimal mostrou\nmaiores adesões pélvicas.\nConclusão O uso de células-tronco mesenquimais na endometriose contribuiu\nnegativamente para a fertilidade, sugerindo o papel dessas células no desenvolvimento\nda doença.\nPalavras-chave\n► endometriose\n► célula-tronco\nmesenquimal\n► fertilidade\n► coelhos\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al.218\n\n\nThe surgical procedures and the extraction and cultivation\nof MSCs were performed at the Operative Technique and\nExperimental Surgery Laboratory and the Cell Therapy Labora-\ntory, both located at the same university in southern Brazil.\nAll handling of the animals followed the rules of the\nCouncil for International Organization of Medical Sciences\n(CIOMS), the Ethical Code for Animal Experimentation\n21 and\nthe precepts of the Colégio Brasileiro de Experimentação\nAnimal (COBEA, www.cobea.org.br).\nThe rabbits were divided into three groups of nine each:\ncontrol group (C), endometriosis group (E), and mesenchy-\nmal group (M). In the endometriosis group, a technique\ndeveloped by do Amaral et al was performed:\n19 am e d i a n\nincision was initially made in the abdominal wall. Then, a\n4-cm piece was removed from the right uterine horn. Patches\n5 /C2 5 mm in size were created and sutured in the retro-\ncervical region.\nIn the control group, a fragment of the uterine horn was\nremoved following the procedure described before. How-\never, the endometrial implant was not sutured in these\nanimals. The mesenchymal group was submitted to the\nsame procedure as the endometriosis group, and later, also\nsubmitted to the application of mesenchymal cells.\nIn all three groups there was a delay of 21 days before\ncopulation was initiated. This is the minimum time required\nfor disease development to occur or for the implanted\nendometrium to become secretory. In the Endometriosis\nand Control groups the copulations occurred after the first\nsurgery. Alredy in the Mesenchymal group, the copulation\nonly started after 21 days of the second surgical procedure\n(application of the mesenchymal cells).\nCopulation occurred 4, 6, and 9 weeks after surgery, when\nthe rabbits were fertile. To check fertility, we evaluated the\ncoloring of the vulva daily, and females with red/violet vulva\n(unlike the normal whitish color)\n23 were placed in a cage\nwith a male for mating. Abdominopelvic ultrasounds were\nperformed, and the animals were then checked for signs of\npregnancy (the presence of a gestational sac or fetus).\n►Fig. 1\nshows a flowchart with a brief description of the chronology\nof the experiments in the study.\nMesenchymal stem cells were obtained 12 weeks before\nthe application by extracting bone marrow from the long\nbones of two rabbits. The femur and tibia were removed, and\nall tissue was scraped away from the bones. The bones were\nplaced in a Petri dish containing phosphate buffered saline\n(PBS; Gibco Invitrogen, NY, USA). The epiphyses were cut to\nexpose the medullary cavity. The marrow was removed from\neach bone with flushing medium into a tube (TPP, Trasadin-\ngen, Switzerland) using a syringe with a needle.\nThe collected bone marrow was processed using a\nFicoll–Hypaque density gradient (d ¼ 1.077 g/mL; Sigma\nChemical, St Louis, USA).\n24 The isolated mononuclear cells\nwere plated in 75-cm 2 flasks (TPP), and were cultivated\nusing IMDM (Iscove ’sM o d ified Dulbecco ’s Media; Gibco\nInvitrogen) medium containing 15% fetal calf serum (FCS;\nGibco Invitrogen) and 1% antibiotics. The cultures were\nmaintained in an incubator, and the culture medium was\nchanged every 72 hours. Upon reaching 80% con fluence, the\ncells were trypsinized using a trypsin –EDTA solution\n(Gibco Invitrogen), and were subcultured to obtain the\nrequired number of cells for potency tests and\ntransplantation.\nFig. 1 Chronology of the experiments in the study.\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al. 219\n\n\nThe MSCs were assessed for potency by inducing their\ndifferentiation into adipocytes, osteoblasts, and chondro-\ncytes ( ►Fig. 2 A-F ). For adipogenic and osteogenic differen-\ntiation, cells were seeded on glass coverslips (Sarstedt,\nNewton, USA) in 24-well plates. A commercial differentiation\nmedium (Gibco Invitrogen) was used for 21 days. The MSCs\nwere stained with Oil Red O to analyze adipogenic induction.\nLarge, rounded cells with cytoplasmic lipid-rich vacuoles\nwere observed. Osteogenic differentiation was assessed by\nvisualizing the mineralization of the extracellular matrix\nwith Alizarin Red S staining.\nCells were grown in a micromass culture to promote\nchondrogenic differentiation.\n25 In the chondrogenic differen-\ntiation assays, high-density micromass MSC cultures gener-\nated cellular nodules, which produced large amounts of\ncartilage-related extracellular matrix molecules, such as col-\nlagen. Paraffin sections of the aggregates stained with tolui-\ndine blue showed condensed structures with cuboidal cells\nand chondrocyte-like lacunae.\nUntreated control cultures, which were grown in regular\nmedium without adipogenic, osteogenic, or chondrogenic\ndifferentiation stimuli, did not exhibit spontaneous adipo-\ncyte, osteoblast, or chondrocyte formation after 21 days of\nMSC cultivation.\nAfter 21 days, in the Mesenchymal group, the second\nsurgery for the application of the mesenchymal cells was\nalso performed.\nDuring this reoperation, the size of the lesion formed by\nthe implant was checked. All the implants were considered\nviable (with an area not exceeding 25 cm\n2). We used 2 /C2 106\ncells in each animal (2 ml in a syringe), which were applied\non the edge of the implant.\nFor the histological analysis, endometrial implants fixed in\nformalin were prepared in paraffin blocks. These samples were\nlater sectioned at a thickness of 5 μm (4 cuts per sample),\nstained with hematoxylin and eosin, and evaluated with an\noptical microscope. The lesions were classi fied according to\nKeenan’si n d e x .\n26 Scores were assigned as follows: score of 3: a\nwell-preserved epithelial layer; score of 2: moderately pre-\nserved epithelium with leukocyte in filtration; score of 1:\npoorly preserved epithelium (occasional epithelial cells\nonly); and score of 0: no epithelium (\n►Fig. 3 A-B ).\nFig. 2 (A-F ) Differentiation of mesenchymal cells. ( B) The differentiation into the adipocyte line age was demonstrated by staining with Oil Red\nO; ( D) Alizarin Red S staining shows minerali zation of the extracellular matrix in the osteogenic differentiation; and ( F) toluidine blue shows the\ndeposition of proteoglycans and lacunae in the chondrogenic differentiation. ( A, C, E ) Untreated control cultures without adipogenic,\nosteogenic or chondrogenic differentiation stimuli are shown.\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al.220\n\n\nThe sample size was based on similarity with other\narticles; to control Type II Error in the applied tests, test\npower was used.\nQualitative variables were described as frequencies and\npercentages, and quantitative variables were described as\nmeans, medians, and quartiles. Fisher ’s exact test was per-\nformed to compare dichotomous qualitative variables among\nthe groups. The quantitative variables were compared by the\nnonparametric Mann-Whitney and Kruskal-Wallis tests. The\nMann-Whitney test was used for post-hoc multiple compar-\nison with Boniferroni correction. Values of p < 0.05 were\nconsidered statistically signi ficant. The data were analyzed\nwith the Statistical Package for the Social Sciences (SPSS; IBM\nSPSS, Chicago, IL, US) software, version 20.0.\nResults\nThe mesenchymal group had the worst fertility rate (11.1%;\np ¼ 0.015; test power: 84.29%); only one animal in this group\nbecame pregnant. In contrast, seven rabbits in the control group\nand seven in the endometriosis group (77.8%) became pregnant.\nAfter having executed the surgical procedures and waited\n21 days, the time to start fertility was different between the\ngroups. The mesenchymal group had the longest delay when\ncompared with the other groups ( p ¼ 0.005; test power:\n92.24%) (\n►Table 1 ). Considering a signi ficant difference\nfound among the groups, they were compared two by two.\nThe p values and the test power of these comparisons are\nshown in the ►Table 2 .\nThe endometriosis group had 2 fetal deaths (28.57%) and 5\nlive births (71.43%). The control group had 7 pregnancies,\nwhich resulted in 2 fetal deaths (28.57%), 3 abortions\n(42.86%), and 2 live births (28.57%).\nThere was no difference in implant size between the\nendometriosis (median ¼ 55, 1st quartile ¼ 15, and 3rd\nquartile ¼ 90) and mesenchymal groups (median ¼ 55, 1st\nquartile ¼ 25, and 3rd quartile ¼ 80; p ¼ 0.863).\nMacroscopically, the formation of a large adhesion in the\ncavity was noted in the mesenchymal group, even making it\ndiffic u l tt oi d e n t i f yt h ei m p l a n t s(\n►Fig. 4A ). Comparatively,\na minor amount of adhesion was noticed in the endome-\ntriosis group ( ►Fig. 4B ). Finality, discrete adhesion forma-\ntion was observed in the control group. Despite the\nformation of adhesions, endometriotic lesions were only\nfound in the sites implanted, and no dissemination was\nobserved.\nThe persistence of epithelial cells in the endometrial\nimplants was assessed semi-quantitatively with Keenan ’s\nindex\n26 (►Fig. 3 A-B ). The following results were observed\namong the nine analyzed rabbits in each group. The endo-\nmetriosis group included 3 rabbits with a Keenan index of 0,\nand 6 with a Keenan index of 3, and the mesenchymal group\nincluded 2 rabbits with a Keenan index of 0 and 7 with a\nKeenan index of 3 ( p ¼ 0.730).\nFig. 3 (A) Histological image of the endometrial implant with a Keenan index of 3. ( B) Histological picture of the endometrial implant with a\nKeenan index of 0.\nTable 1 Comparison of time until the onset of the fertile period\n(in days) in the three groups\nGroups n Span in days\nEndometriosis 9 33 [18 –34]\nMesenchymal 9 14 [14 –16]\nControl 9 40 [33 –40]\nNote: Nonparametric test of Kruskal-Wallis; p < 0.05; median [1 st -3 rd\nquartiles]; Test power ¼ 92.24%.\nTable 2 Test power and p values in the inter-group comparison\nof time until the onset of the fertile period\nGroups p Test power\nControl x Endometriosis 0.061 0.3314747\nControl x Mesenchymal 0.043 0.7495601\nEndometriosis x Mesenchymal < 0.001 0.9583571\nNote: Nonparametric test of Kruskal-Wallis; p < 0,05.\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al. 221\n\n\nDiscussion\nThe selection of rabbits as an experimental model was based\non their ef ficacy for developing ectopic endometrial foci in\nthe abdominal wall. 8 It is known that the production of\nvarious cytokines occurs in endometriosis, which generally\nstimulates endometrial proliferation and contributes to the\nproduction of acute-phase in flammatory proteins, creating a\npro-inflammatory environment.\n22 It is also known that in\nendometriosis, the endometrial cells exhibit the character-\nistics of chronicity and expansion, showing capacities for\nmigration, invasion, and apoptosis resistance. 9\nThis scenario has led to new theories exploring the\ninvolvement of the immune system, such as immunological\nmechanisms facilitating the development of endometriosis,\nor the immune system being activated by it. 9 The involve-\nment of stem cells (particularly of the mesenchymal lineage)\nin the origin of the disease has also been proposed.\n11,12,15 In\ncontrast with the reports on endometriosis leading to in-\nfertility,6,27 the development of endometriosis in the rabbits\ndid not worsen the fertility rate. However, the rabbits were\nnot a good model for endometriosis-related infertility. The\nuse of the obtained MSCs caused the relative risk of infertility\nto increase 4-fold in these animals.\nOne mechanism that may explain the aforementioned\nresult involves the pathophysiology of the disease. The\nevidence of stem cells in the human endometrium and the\nsuggestion of the presence of stem cells —specifically the\nmesenchymal lineage\n8—may be related to the worsening of\nthe fertility rate in the group that received mesenchymal\ncells, because we used cells that may be involved in the origin\nand progression of the disease.\nAnother mechanism that may be involved is the epithe-\nlial–mesenchymal transition (a process comprising a series\nof changes in epithelial cell phenotype that causes epithelial\ncells to assume the characteristics of MSCs, such as having a\ngreater capacity for migration, invasion, and apoptosis\nresistance).\n28 Because endometriotic cells in the context of\nendometriosis also take on this function, 8,9,28,29 they repre-\nsent a sum of the effects of both mechanisms.\nA question remains regarding whether the mesenchymal\ngroup’s delay in entering the fertile period was related to the\ninvolvement of MSCs in the origin and progression of\nendometriosis.\nWith regard to obstetric outcomes, the endometriosis and\ncontrol groups showed no statistically signi ficant difference.\nThis result may be because of the small sample size. The\nmesenchymal group requires further analysis because of its\nlow fertility rate.\nThe macroscopic evaluation of the pelvic cavity of each\nanimal (during euthanasia) revealed that the mesenchymal\ngroup had a greater rate of adhesions than the other groups,\nin an analogy with the widely used Revised Classi fication of\nthe American Society for Reproductive Medicine (ASRM).\n30\nThus, we could consider the mesenchymal group a carrier of\nstage-IV disease (severe endometriosis with dense and firm\nadhesions).\nIt is known that anatomical distortion and adhesions are\nsome of the mechanisms that generate infertility in the\ncontext of endometriosis.4,21,31 This mechanism may explain\nwhy the group that received MSCs on the endometrial\nimplants may have had a low fertility rate: its higher rate\nof pelvic adhesions.\nFinally, a histological analysis of the implants using Keen-\nan’s index was performed. This index measures the persis-\ntence of epithelial cells in endometrial implants.\n26 A\ncomparison of the two groups revealed no statistically sig-\nnificant difference in the scores of Keenan ’s index.\nAlthough it may be possible that the worsening of fertility in\nthe mesenchymal group can be explained by two other\nmechanisms (the longer time the animals were exposed to\nthe implants and the occurrence of two surgical procedures in\nthis group), in this study design, according to our results, we\ncan conclude that applying MSCs on female rabbits via en-\ndometrial implants provides a substrate for the development of\nendometriosis and thus harms fertility in these animals.\nFig. 4 (A) Mesenchymal group: large presence of adhesions (yellow arrows) that made it dif ficult for the implant to adhere (white arrows); ( B)\nEndometriosis group: presence of a minor amount of adh esion (yellow arrow) next to the implant (white arrow).\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al.222\n\n\nDeclaration of Con flicting Interests\nThe Authors have no con flicts of interest to declare.\nAcknowledgments\nWe would like to thank the teams from Vivarium, from the\nOperative Technique Laboratories, and from the Cell\nTherapy and Pathology Departments of Pontifícia Univer-\nsidade Católica do Paraná (PUC -PR) for their dedication and\nsupport.\nReferences\n1 Burney RO, Giudice LC. 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Stem Cells Int\n2016;2016:3573846\n30 Revised American Society for Reproductive Medicine Classi fica-\ntion of Endometriosis. Fertil Steril 1996;67(05):817 –821\n31 de Ziegler D, Borghese B, Chapron C. Endometriosis and inferti-\nlity: pathophysiology and management. Lancet 2010;376(9742):\n730–738\nRev Bras Ginecol Obstet Vol. 39 No. 5/2017\nThe Effect of MSCs on Fertility in Experimental Retrocervical Endometriosis Abreu et al. 223","source_license":"CC0","license_restricted":false}