{"paper_id":"1e95ce51-191e-43ec-bf29-64295f30013d","body_text":"ORIGINAL ARTICLE\nMorphology of human endometrial explants and secretion\nof stromal marker proteins in short- and long-term cultures\nNick A. Bersinger & E. M. Genewein & O. Müller &\nH. J. Altermatt & B. McKinnon & M. D. Mueller\nReceived: 9 July 2009 / Accepted: 28 August 2009 / Published online: 18 September 2009\n# Springer-V erlag 2009\nAbstract Human endometrial tissue is frequently biopsied\nunder surgical and laparoscopic procedures for the inves-\ntigation of infertility, abdominal, or menstrual pain. These\nsymptoms often but not always are the consequence of\nendometriosis, which is characterised by the growth of\nendometrial tissue outside the uterine cavity and affecting\n8–10% of women during the fertile age. First-line treatment\nis often by surgery. Biopsied endometrial tissue is not only\nused for immunohistochemical examination but has also\nbeen cultured in vitro. Explant culture systems maintain the\nthree-dimensional structure of the tissue, but so far no\nmorphological validation studies are available for the\nstromal cells which are responsible for the production of\nhormones and inflammatory cytokines in the endometrium.\nWe have documented, by transmission electron microscopy,\nthe morphological alterations of stromal cells in short-\n(12 h) and long-term (7 days) cultures of endometrial\nexplants biopsied in the postovulatory phase. The produc-\ntion of prolactin, a stromal cell marker, was determined. We\nfound that the morphological integrity of these cells was\nstarting to be disrupted from as early as 12 h in culture.\nSome stromal cells, however, developed into predecidual\ncells. After 96 h, a large fraction of the cell population was\nnecrotic, and after 7 days, the cytoplasm had disappeared.\nIn presence of progesterone, the decay of stromal cell\nintegrity was slowed down. The release of prolactin and\nIGF-binding protein-1 during culture followed the morpho-\nlogical pattern. We conclude that the explant culture model\nis viable for not more than 48 h in vitro for stromal cells,\nbut that this interval can be prolonged by the addition of\nprogesterone which initiates decidualisation.\nKeywords Endometrium . Stromal cells . Explant culture .\nTransmission electron microscopy\nIntroduction\nSurgically removed endometrial explants are often used\nculture systems for investigations in gynaecologic endocri-\nnology and immunology. These systems maintain their\nthree-dimensional structure and are competent for the de\nnovo synthesis of endometrial proteins. The morphological\nvalidation for epithelial cells in explants cultured (for more\nthan 4 days) has been provided by Dudley et al. [ 1], but no\nsuch validation has been given on stromal cells neither for\nmorphological criteria nor for the specific production of\nbiochemical markers.\nThe aim of this study was the documentation of\nmorphological alterations of stromal cells and the analysis\nof the correlation between these and the secretion of\nN. A. Bersinger : E. M. Genewein : M. D. Mueller\nDepartment of Obstetrics and Gynaecology, University of Berne,\nBerne, Switzerland\nO. Müller\nDepartment of Anatomy, University of Berne,\nBerne, Switzerland\nH. J. Altermatt\nDepartment of Pathology, University of Berne,\nBerne, Switzerland\nB. McKinnon\nDepartment of Clinical Research, University of Berne,\nBerne, Switzerland\nN. A. Bersinger ( *)\nUniversity of Berne, Endometriosis and Reproductive Medicine,\nDKF Murtenstrasse 35,\nCH-3010 Berne, Switzerland\ne-mail: nick.bersinger@dkf.unibe.ch\nGynecol Surg (2010) 7:75 –80\nDOI 10.1007/s10397-009-0520-4\n\nstromal marker proteins prolactin (PRL) [ 2, 3] and insulin-\nlike growth factor-binding protein-1 (IGF-bp1) [ 4, 5]i n\nshort-term (12 h) and long-term (up to 7 days) cultures of\nendometrial explants of the postovulatory phase. Short- and\nlong-term cultures as defined by the time intervals given\nabove were assessed by light and electron microscopy.\nMaterials and methods\nThis project focused on the morphology of endometrial\nexplants after culture; the major endpoint of the study was\nthe transmission electron microscopic aspect of the epithe-\nlial and particularly, the stromal cells at scheduled times\n(after 12, 24, 48, 96, and 168 h in culture). The production\nof the stromal marker proteins prolactin and IGF-bp1 was\ndetermined as an accompanying measure only.\nProgesterone-stimulated cultures were run as positive and\nnegative controls, respectively.\nEndometrial tissues from three nulliparous women were\ntaken, in the postovulatory phase of a normal cycle, after\nhysterectomy performed for benign reasons. Absence of\nendometriosis was demonstrated histologically, no hormon-\nal treatment was given, and informed consent was obtained\nfrom the patients. Preconditions were known infertility,\nregular cycle, and no substitution of hormones. The cycle\nphase and ovulation were ascertained by the determination\nof progesterone in a serum collected at the time of surgery.\nThe tissue was dated according to the criteria of Noyes [ 6].\nFragments of 2 –3m m\n3 were cultured in 2 mL Dulbecco's\nmodified Eagle's medium (DMEM) with added glutamine\nand antibiotic/antimycotic, both to 1% v/v from stock\nsolutions (GIBCO-Invitrogen, Paisley, Scotland) under 5%\nCO2 in air. After 12, 24, 48, 96, and 168 h, an aliquot of\nexplant was examined by light microscopy after fixation in\n100% ethanol and by transmission electron microscopy\nafter fixation in 3% glutaraldehyde. At the same time, an\naliquot of the conditioned supernatant was removed and\nstored at −30°C for the subsequent determination of the\nstromal cell-specific markers prolactin by dissociation-\nenhanced lanthanide fluoroimmunoassay (Delfia®, Wallac\nOy, Finland) and of IGF-bp1 by a microplate enzyme\nimmunoassay purchased from Bioserv, Rostock, Germany.\nPositive stimulation control cultures were run in the\npresence of progesterone (200 nM).\nResults\nEpithelial cells\nThe morphological configuration of epithelial cells (cell\nlayer with microvilli, ciliae, and nucleus) was found to\nremain intact until 96 h in culture. Thereafter, apoptotic\nalterations in the nucleus and cytoplasm were observed.\nLipid granula were seen early after the setting up of the\nculture. This is shown in Fig. 1 (a: before culture; b, c: after\n24 and 96 h in culture, respectively).\nFig. 1 Endometrial epithelial cells.a Intact cell at the time of sampling.\nCellular borders are clearly visible, but the cells are connected through\ninterdigitations (I) and desmosomes ( D). N nucleus, Nl nucleolus, Mi\nmitochondria, rER endoplasmatic reticulum, G Golgi apparatus, Gly\nsmall deposits of glycogen, Bm basement membrane, S stromal cell. b\nAppearance of lipid granula in epithelial cells after 24 h in culture. Mv\nmicrovilli, Chol crystallised cholesterol. c Epithelial cell with cilia ( Kz)\nafter 96 h in culture. AV autophagic vacuole, Ly lysosomes, L lipid\ndeposit. Magnification, ×7,650\n76 Gynecol Surg (2010) 7:75 –80\n\nStromal cells\nCulture without supplements\nThe morphological integrity of stromal cells (Fig. 2a) was\ndisrupted early; already after 12 h of culture dilatation of\nrough endoplasmatic reticulum, condensation of nuclear\nchromatin and the appearance of apoptotic vesicles were\nnoted (Fig. 2b). Nevertheless, some stromal cells were\nfound to develop into predecidual cells as identified by the\npresence of glycogen storage vesicles and lipid droplets, an\nincreased cytoplasmic volume and a higher number of cell\ncontacts (Fig. 2c). After 96 h of culture, glycogen storage\nvesicles are still visible (Fig. 2d), but a large fraction of the\nstromal cell population was necrotic. After 7 days in\nculture, however, the stromal cells were degenerated by\nnecrotic and apoptotic events, and the cytoplasm had\ndisappeared (Fig. 2e). At this stage, the epithelial cells\nwere still in contact with each other, but this was beginning\nto loosen, and the number and size of the lipid droplets had\nincreased. This is shown in Fig. 3 which was taken at the\nintersection of the two cell types but with partial disap-\npearance of the basal membrane.\nThe release rate of the stromal markers into the medium\nduring culture followed the morphological pattern. Although\nprolactin concentration increased in the supernatant up to\nFig. 2 Endometrial stromal cells. a Intact cell at the time of sampling.\nThe cell membrane is intact but intercellular contact is scarce.\nOccasional granula of unknown origin, but no lysosomes or lipid\ndeposits can be seen. b Stromal cells after 12 h in culture. First signs\nof degenerative processes can be observed. Nuclear invaginations and\nchromatin densification at the borders ( arrows) have increased, and\nthe matrix is partially dissolved. Lysosomes ( Ly) and lipid deposits\n(Lk) are visible. c Stromal cells after 48 h in culture. V acuoles ( V) and\nglycogen deposits have increased. Nucleoli ( Nl) are prominent and\nstructured. d Stromal cells after 96 h in culture. Large glycogen\ndeposits are signs of decidualisation in the surviving cells, while the\nmajority of cells is necrotic or apoptotic ( Z). e Stromal cells after\n7 days in culture. Large scale degeneration through apoptotic and\nnecrotic processes. The cytoplasm ( C) has dissolved, and apoptotic\nbodies are separating from the cells. The cells at the bottom right (E)\nare epithelial cells with glycogen inclusions. Magnification, ×3,550\nGynecol Surg (2010) 7:75 –80 77\n\n96 h in culture (Fig. 4a), the production rate per unit time of\nthe hormone was highest after 12 h (4.07 ± 2.00 pg/mL/h,\nmean ± standard deviation) and decreasing thereafter\n(3.14 ± 1.37 pg/mL/h, N=6, Fig. 4b). The release rate of\nIGF-bp1 (Fig. 4d) peaked between 24 (14.4 ± 8.0 pg/mL/h)\nand 48 h (14.0 ± 9.7 ng/mL/h, N=4) and similarly declined\nthereafter.\nCulture in presence of progesterone\nThe addition of progesterone results in the in vitro\ndecidualisation of endometrial stromal cells. We have added\nprogesterone (200 μM) to our cultures and found that the\ndecay of the stromal cell tissue seen above was consider-\nably slowed down. Figure 5a shows stromal cells under\ndecidualisation after 7 days in culture with progesterone.\nAfter this incubation period, epithelial cells also seem to be\nin better contact with each other, through desmosomes and\ninterdigitations, in presence of the hormone (Fig. 5b) than\nin its absence (Fig. 3), and the basal membrane is present.\nThis protection of the morphological features by progester-\none was due to an inhibition of apoptosis as indicated by a\ndecrease in terminal deoxynucleotidyl transferase-mediated\ndUTP nick end labelling (TUNEL) signals (not shown).\nIn presence of progesterone, the release of prolactin\n(Fig. 4c) and IGF-bp1 (Fig. 4d) was strongly increased.\nPeak production rates per unit time were reached after 48\nand 96 h in culture for prolactin and IGF-bp1, respectively,\nFig. 3 Epithelial and stromal cells after 7 days in culture. Epithelial\ncells make up the top half of the panel (arrow at right ); they are still\nloosely associated but the connexions between them ( I, D, as shown in\nFig. 1a) have become scarce. Lipid inclusions ( L) and lysosomal\nremains (LR) have further increased in size. The basal membrane ( Bm,\nseparating the two cell types through the middle of the panel ) is only\npartially visible. The bottom half of the panes (arrow at right ) shows\ndegerated stromal cells, with condensed chromatin ( kC), phagosomes\n(Ph), and lysosomal remains. Magnification ×7,650\nFig. 4 Marker production by\ncultured endometrial stromal\ncells. a Concentration\n(accumulation) of PRL in the\nsupernatant; six cultures, each\nin duplicate. b PRL production\nrate per hour, showing the\nconstant decrease with time in\nculture. c PRL concentration\n(accumulation) in presence of\nprogesterone (200 ng/mL); note\nthe difference in scale compared\nto the control experiment with-\nout progesterone ( a). d IGF-bp1\nproduction by cultured endome-\ntrial stromal cells. The graph\nshows the concentration\n(accumulation) of IGF-bp1 in\nthe supernatant (two cultures,\neach in duplicate). No\nprogesterone added ( open\ncircles), culture in presence\nof progesterone ( closed circles ,\n200 nM)\n78 Gynecol Surg (2010) 7:75 –80\n\nand their concentrations in the supernatant continued to rise\nafter 7 days in culture (Fig. 4c, note the difference in scale\nwhen comparing to the control, Fig. 4a).\nDiscussion\nOur observations concerning the morphology of epithelial\ncells correspond to the published validation study [ 1] where\nthe viability of these cells over 4 days was documented.\nLater in the course, apoptotic features, like the condensation\nof nuclear chromatin and autophagy, are seen. The occur-\nrence of lysosomes, glycogen, and lipid bodies increased\nduring culture time, which is meant to be a sign of\ndegeneration. On the other hand, we detected an increased\nnumber of lipid granules in epithelial cells stimulated by\nprogesterone. According to Dallenbach [ 7], this feature\nseems to be a sign for increased cell activity; the epithelial\ncells stay intact for a longer time in culture than stromal cells\ndo. The production of prolactin and IGF-bp1 similarly rises\nafter the addition of progesterone. At the beginning of the\nculture, proteins are secreted from apoptotic vesicles, as well\nas synthesised de novo by the cells, and accumulate in\nsupernatant. After 48 and 96 h, prolactin and IGF-bp1 are\ndestroyed by proteases released by necrotic cells. In spite of\nthis consideration, further studies, e.g., with purified epithe-\nlial cells in primary culture, are required to clarify whether\nepithelial markers (e.g., glycodelin) also increase in vitro\nwith the addition of progesterone and whether the develop-\nment of lipid bodies due to progesterone is a truly stromal\ncell-mediated event. Stromal cells seem to be severely\naffected morphologically during the early culture phase by\napoptotic features and later by excessive necrosis. The\nproduction of prolactin declined, probably due to proteases\nsecreted by necrotic cells at the same time. Studies with a\nprotease inhibitor should clarify this consideration. Despite\nthe overall degenerative state, a fraction of stromal cells\nseems to develop into predecidual cells without the addition\nof progesterone. To our knowledge, this has not been\ndescribed to date. The development of decidual cells during\npregnancy (i.e., exposure to high progesterone levels),\nhowever, has been studied, and the induction of decidualisa-\ntion of stromal cells, together with increased prolactin\nproduction, by progesterone was confirmed [ 4, 8–11]. A\nsingle short application of progesterone induces development\nof stromal cells to decidual cells [ 12]. In this study, we have\nidentified areas with decidualised stromal cells in\nprogesterone-added culture. Using light and transmission\nelectron microscopy together with the TUNEL method, we\ncould show that the tissue is protected in presence of\nprogesterone and that stromal cells remain morphologically\nintact for an extended time. The inhibition of apoptosis by\nprogesterone has been demonstrated previously [ 13]. Based\non our observations, stromal cells are viable for 7 days in\nculture in presence of progesterone at 200 ng/mL.\nIn conclusion, we have observed in this preliminary study\nthat, in contrast to the validation study of Dudley [ 1], the\nendometrial explant system is limited by an early decay of\nstromal cells in the absence of added progesterone. Products\nof stromal cells, like prolactin and IGF-bp1, are detected in\nthe supernatant of endometrial explant cultures, which could\nbe due at least in part to early cell decay and the release of\nFig. 5 Morphology of stromal\nand epithelial endometrial cells\nafter culture in presence of\nprogesterone (200 nM). a\nDecidualised stromal cells after\n7 days in culture in presence of\n200 nM progesterone. Well-\npreserved cells with normally\nconfigured nuclei ( N) containing\nglycogen and lipid deposits.\nMagnification, ×7,600. b\nEpithelial cells after 7 days in\nculture in presence of 200 nM\nprogesterone. In contrast to\nthe control culture without the\nsteroid hormone (Fig. 3), the\nintercellular contacts are still\nwell established even after the\nlong culture period. For\nabbreviations, see legend to\nFig. 1. Magnification, ×13,400\nGynecol Surg (2010) 7:75 –80 79\n\npreviously synthesised markers. However, as long as stromal\ncells show apoptotic features, the culture system is viable,\nand this seems to be the case for 48 h. This interval can be\nprolonged by the addition of progesterone, which not only\nslows stromal cell decay but also stimulates the decidualisa-\ntion of these cells. In the future and in an extension of this\npreliminary project, it would be interesting to compare the\nmorphological observations in endometrial tissue from\nhealthy (besides infertility), cycling women cultured under\ncontrol, not oestrogen stimulated conditions (as done in this\nstudy) between different stages of the menstrual cycle, or\nbetween the absence or presence of oestrogen (17β-estradiol)\nin the culture. Moreover, as evidence is increasing that, in\nwomen suffering from endometriosis, there are significant\ndifferences between cases and healthy controls even in the\neutopic (intrauterine) tissue, the project could be extended to\nthe comparison of electron microscopic features as a function\nof the severity of endometriosis. This would, however,\nrequire the creation of different groups of patients and\ntherefore, result in a large number of cultures and EM\nanalyses to be performed and statistically analysed.\nConflict of interest There is no actual or potential conflict of\ninterest in relation to this article.\nReferences\n1. Dudley DJ, Hatasaka HH, Branch DW, Hammond E, Mitchell\nMD (1992) A human endometrial explant system: validation and\npotential applications. Am J Obstet Gynecol 167:1774 –1780\n2. Riddick DH, Luciano AA, Kusmick WF, Maslar IA (1978) De\nnovo synthesis of prolactin by human decidua. Life Sci 23:1913 –\n1922\n3. 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