{"paper_id":"3d14a5ad-42dd-4732-94e4-ec5c1749fea9","body_text":"~ 199 ~ \nInternational Journal of Clinical Obstetrics and Gynaecology 2019; 3(1): 199-204 \n \nISSN (P): 2522-6614 \nISSN (E): 2522-6622 \n© Gynaecology Journal \nwww.gynaecologyjournal.com \n2019; 3(1): 199-204 \nReceived: 17-11-2018 \nAccepted: 23-12-2018 \n \nDr. Roya Rozati \nM.B.B.S., M.D., F.R.C.O.G., \nProfessor and Head, Department \nof Obstetrics and Gynaecology, \nOwaisi Hospital and Research \nCentre; Medical and Research \nDirector, M.H.R.T, Banjara Hills, \nHyderabad, Telangana, India \n \nWajeeda Tabasum \nMaternal Health and Research \nTrust, Telangana, India \n \nAyapati Gautam Mehdi \nMaternal Health and Research \nTrust, Telangana, India \n \nAyapati Vikram Aiman \nMaternal Health and Research \nTrust, Telangana, India \n \nDr. Avinash Bhadria \nOwaisi Hospital and Research \nCentre, Telangana, India \n \nDr. Sumaya Fatima \nPharm-D, Department of \nPharmacy Practice, Deccan School \nof Pharmacy, Owaisi Hospital and \nResearch Centre, Hyderabad, \nTelangana, India \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nCorrespondence \nDr. Roya Rozati \ndrroyarozati@gmail.com \nM.B.B.S., M.D., F.R.C.O.G., \nProfessor and Head, Department \nof Obstetrics and Gynaecology, \nOwaisi Hospital and Research \nCentre; Medical and Research \nDirector, M.H.R.T, Banjara Hills, \nHyderabad, Telangana, India \n \nIdentification of cells without colony-forming ability in \nEutopic endometrial progenitor cells in infertile women \nwith endometriosis in comparison with healthy fertile \nwomen in Indian perspective \n \nDr. Roya Rozati, Wajeeda Tabasum, Ayapati Gautam Mehdi, Ayapati \nVikram Aiman, Dr. Avinash Bhadria and Dr. Sumaya Fatima \n \nDOI: https://doi.org/10.33545/gynae.2019.v3.i1d.33 \n \nAbstract \nEndometriosis is a chronic benign gynecological disease characterized by the presence of  ectopic \nendometrial tissue outside of the uterus cavity. Human Endometrium is a highly regenerative tissue . The \naim of our study is to screen potential stem cell markers for the prospective isolation of human endometrial \nstromal stem/progenitor cells . To determine their capacity to identify colony forming in eutopic \nendometrial progenitor Cells in infertile Women and healthy Women with Endometriosis in Indian \nperspective. This study identified CD90 marker of colony -forming human endometrial stromal cells a re \nfound to be positively stained. \n \nKeywords: Endometrium, progenitor cells, endometriosis, clonogenicity, infertility \n \n1. Introduction  \nEndometriosis is chronic benign gynecological disorder which is characterized by a growth of \nendometrial tissue outside the uterine cavity. Human endometrium undergoes cyclical processes \nof growth, differentiation, shedding, and regeneration as part of the menstrual cycle during the \nreproductive life of women (Maruyama T et al. 2008) [24]. Estimates shows that endometriosi s \naffects 10-15% of all women population during reproductive age with pelvic pain and infertility \n(Sasson IE et al . 2008, Allaire C  2006) [32, 2] It is frequently associated with dysmenorrhea, \nmenorrhagia and dyspareunia, leading to infertility (Surrey ES 2003, Oral E et al. 1997) [34, 29] \nThe pathological condition involves adhesion, proliferation, and development of the endometrial \nimplants in ectopic regions such as the ovary and the peritoneal cavity.  The pathogenesis of \nendometriosis is ascertained but  there are 3 different entities which are involved in \nendometriosis are ovarian, peritoneal endometriosis and deep endometriotic nodules (Nisolle M \net al . 1997)  [28]. There are evidence which indicates that eutopic endometrium in women \nsuffering from endometriosis is different from that of healthy controls. Apart from contributing \nfactors like genetic predisposition, environmental factors, hormonal, alterations immune and \nendocrine functions plays a crucial role in  the pathogenesis and etiology of endometri osis \n(Bondza P.K et al. 2009, Carvalho, L 2011 and Jensen, J.R  2010) [3, 4, 21 ]. The knowledge about \nthe etiology and pathogenesis of this disease still remain uncertain, but there are a number of \nleading theories including retrograde menstruation, altered  immunity, coelomic metaplasia, and \nmetastatic spread. \nEndometriosis is an estrogen -dependent benign inflammatory disease characterized by the \npresence of ectopic endometrium (Giudice et al. 2004) [18]. The role of eutopic endometrium in \nendometriosis-related infertility is still unclear due to a lack of understanding about the normal \nphysiologic mechanisms. The eutopic endometrial glandular and Stromal cells may be \nfunctioning differently in women with endometriosis compared to normal women. These cells \nhave characteristics which have favored the survival outside the uterine cavity and precede \ndevelopment of well -documented changes at the peritoneum and other ectopic sites  (Akoum A \net al . 2006)  [1]. During menstruation, the endometrial cells in endometriosi s patients could \nescape immune surveillance from the body and are less susceptible to apoptosis, resulting in an \nincrease in viable cells.  After overcoming a phase of immune tolerance, the next step in the  \ndevelopment of early endometriosis is the adhesion of endometrial cells to mesothelium and \n\n\nInternational Journal of Clinical Obstetrics and Gynaecology \n~ 200 ~ \ninvasion of the extracellular matrix, since the eutopic \nendometrium of women with endometriosis are more adhesive \nand invasive normal endometrium. After the last step of \nangiogenesis, the endometrial cells establish a new blood supply \nfor the survival of implants, continue to proliferate in ectopic \nsites, and finally results in endometriosis \nIn a regular event, in response to the mounting levels of \nestrogen, new functionalis layer begins to grow during \nProliferative phase  (Ferenczy A et al . 1979)  [11]. Rising \nprogesterone in secretory phase blocks epithelial mitosis, and \ncells undergo differentiation.  \nA striking feature of the human endometrium is spontaneous \ndecidualization of the stromal compartment during the m id-\nluteal phase of each cycle, a process also responsible for the \nmenstrual shedding of the endometrium in the absence of \npregnancy (Lam EW et al 2012) [23]. The colony forming units \nhave self -renewal capacity by undergoing cloning in vitro \n(Gargett, C.E 2007, Morrison, S.J 1997)  [16, 27]. Adult stem cells \nin human endometrium are clonogenic which are idenitified \nfrom small population of colony forming units (CFU). The \nhuman endometrium contain small population of clonogenic \nepithelial (0.22%) and stromal c ells (1.25%), exhibiting stem -\ncell function in vi tro. (chan rw 2004) [6] The clonogeniety of \ncells from proliferative, secretory, and inactive endometrium \nwas demonstrated too. (schwab ke 2005) [33]  \nThe human endometrium contained small populations of \nepithelial progenitor cells and MSC -like cells (Gargett, C.E. \n2009) [13, 14]. Cultured endometrial stromal cells also differentiate \ninto mesodermal lineages and lineages of ectodermal and \nendodermal origin (Wolff, E.  F et al .2007, Wolff E.  F et al . \n2010, Santamaria X et al. 2011, Dimitrov, R.  et al. 2008) [37, 36, \n31, 8 ] indicating that endometrial stromal cells have considerable \nplasticity. \nThe human endometrium exhibits remarkable regenerative \ncapacity (Gargett CE et al . 2012) [12] which is rich in \nmesenchymal stem -like cells (eMSCs), and are immuno -\nprivileged compared to other types of stem -like cells, rendering \nthem a promising resource for cell -based therapies Santamaria, \nX et al. 2011, Wolff, E.F et al. 2011, and Ulrich D et al. 2013) \n[31, 35, 38]. In the absence of implantation, the functionalis relapses \nand sheds during menstruation, commencing a new cycle.  \nRecent studies have been demonstrated that stem - progenitor \ncells play an important role in the onset of gynecological \ndiseases such as endometriosi s (Gargett CE et al. 2008) [15]. In \nstem cells, the human endometrium contains a small population \nof endometrial epithelial and stromal cells with high \nproliferative potential (Chan RW et al. 2004, Gargett CE  et al. \n2005) [6, 17]  \nMesenchymal or stromal st em cells are considered as separate \nstem cell population which (MSC) have several stroma -\ncontaining tissues, including bone marrow, synovial fluid, dental \npulp, adipose tissue, cord blood and skeletal muscle Minguell JJ \net al . 2001, Romanov YA et al . 2003) [26, 30 ]. MSC have the \nability to differentiate into cells of a different phenotype than \ntheir tissue of origin (Herzog EL et al . 2003, Grove JE  et al . \n2004) [20, 19]. Various markers have been used to isolate MSC. \nMarkers which have been used to partiall y purify MSC include \nCD34 and CD90. CD90 is an accepted marker of cultured \nMSC27 and it has never been used to isolate MSC, but rather has \nbeen used in combination with other negative markers. \nThe aims of this study were to screen potential stem cell markers \nfor the prospective isolation of human endometrial stromal \n/progenitor cells, to determine the capacity to identify colony \nForming in eutopic Endometrial progenitor Cells in infertile \nWomen and healthy Women with Endometriosis in Indian \nperspective and the location of cells expressing these markers in \nhuman Endometrium. This study used a colony-forming assay as \na screening test for identifying potential markers of endometrial \nStromal stem/progenitor cells. \n \n2. Materials and Methods \nClinical samples of endometriotic tissues were collected from 30 \npatients from reproductive age group between 18 -35 years were \ncollected at the Maternal Health research Trust and Owaisi \nHospital and Research Centre, Hyderabad, who underwent \nlaparoscopic surgery between Aug 2015  – July2017. Informed \nwritten consent was obtained from each patient as a part of the \nstudy protocol. \nOut of 30 patients 15 were diagnosed with stage1 endometriosis \nwho had not taken exogenous hormones for 3 months prior to \nsurgery were only included . Menstrual cycle stage, assessed by \nhistological examination according to well -established criteria, \nwas obtained from pathology reports.  \n \n3. Methodology \nEndometrial tissue samples were collected in 1 X Phosphate \nbuffered saline with 10% antibiotic anti -mycotic solution. \nSamples were maintained at 4  oC AND processed. All samples \nwere processed within 2 hrs from collection. The tissues were \ndigested with collagenase III (HyClone Laboratories, USA) for \n30 mins at 37 oC. Samples were pipetted vigorously in between . \nThe digestion was stopped by adding absolute media to the \nsamples. All the samples were centrifuged at 100g X 10 mins \nand the pellet was suspended in 2 ml media. All the samples are \ncultured at 37 oC with 5% CO 2 in a humidified incubator. After \nsurgery, endometrial biopsies were fixed in formaldehyde fixed, \nand hematoxylin -stained cross sections were analyzed by \nexperienced histopathologists for assessment of the grade of \nendometriosis (I –IV) and for determination of the stage of the \nmenstrual cycle (prol iferative or secretory), referring to \nestablished histological criteria \n \n3.1 Cell culture \nThe cells were cultured in Dulbecco's modified Eagle's medium \n(DMEM, Gibco) with 1% Antibioticantimycotic solution \n(Gibco), and 15% FBS (HyClone Laboratories). The culture \nmedium was replaced every 48 h. For passaging, the cells were \nwashed with 1X PBS (pH 7.2)  and treated with 0.25% trypsin -\nEDTA (Gibco) for 15 min at 37 oC, complete DMEM was added \nto stop the trypsinisation reaction.  The cells were centrifuged at \n100g X 10 mins and resuspended in culture medium. The cells \nwere cultured till three passages (P3) before clonogenic assay. \n \n3.2. Clonogenic assay \nCultures, out of passage 3 (P3), were plated at a density of 100 \ncells/cm2 in DMEM (Gibco), along with 1% Antibiotic anti -\nmycotic solution (Gibco), and 15% FBS. Colony formation was \nmonitored regularly. On da y 14, cells were fixed with absolute \nmethanol for 2 minutes and stained with 1% crystal violet \naqueous solution for 5 minutes. (Clones or colony -forming units \n(CFUs) consisting of 50 cells were counted to determine the \ncloning efficiency (CE) percentage, w hich was the number of \ncolonies formed per seeded cell multiplied by 100.) Colonies \nwith more than 50 cells were counted for the assay. Each assay \nwas repeated twice, and cloning efficiency [CE] was calculated \nas CE% = (n. clones/cells seeded) X 100.  \n \n \n\nInternational Journal of Clinical Obstetrics and Gynaecology \n~ 201 ~ \n4. Statistical analysis \nColony-forming capacity was performed using GraphPad Prism \n5 (GraphPad Software, Inc., San Diego, CA, USA). Unpaired t-\nTest was performed to evaluate the difference between clonal \nefficiency in women with and without endometriosis.  Data is \npresented as Mean ± SEM. Variable difference with P< 0.05 is \nconsidered statistically significant. The sample size was \ndetermined by using the open  EPi statistics and 95% of \nconfidence was used to detect the results with 90% of sample \npower.  \n \n5. Results \nSmall populations of human endometrial stromal cells expressed \neach of the markers. CD90 was strongly expressed by \nfunctionalis stroma and perivascular cells, but only weakly \nexpressed in the basalis stroma. This study identified CD90 as a \ncandidate marker of colony-forming human endometrial stromal \ncells supporting the concept that human endometrium contains a \npopulation of stromal stem/progenitor cells.CD90  has been \nlinked to spindle shape cells.  Primarily both epithelial and \nStromal cells were culture d. Epithelial cells died around 7 -8 \ndays of culture. After 10 -14 days, the cultures consist of spindle \nlike cells. With each passage, the cell population became more \nhomogenous, and after third passage the spindle like cells  were \nthe only type detected in the culture.  The results clearly \ndemonstrate that endometrial cells are negative for CD34 and \npositively stained for CD90 (fig: 1) \nThe clonogenicity of endometrium from women with \nendometriosis (n = 15) and without endometriosis (n = 15) was \ncompared. The total clonogenic efficiency of endometrial \nprogenitor cells was significantly greater (0.25 ± 0.03 %) in \nfertile women compare with endometriosis (0.13 ± 0.01%, p< \n0.05). The categorization of colony size for endometrial \nprogenitor cells was observed in of both the groups. Small CFUs \nwere defined as comprising <4000 large loosely -packed cells \nand large CFUs as comprising > 4000 cells with a dense center \nof tightly packed cells. During the first week of culture, the \ngrowth rates for the two colony types were similar, with colonies \ngenerally comprising <100 cells after 7 days. Around day 10 to \nday 11, however, small CFUs stopped proliferation and \nmaintained their size, but the growth of some colonies increased \ndramatically and formed large CFUs containing as ma ny as \n15,000 cells by day 14 (Figure 2a,  2b, 2c). There is no \nsignificant difference in the larger colonies between the woman \nwithout endometriosis and with endometriosis (0.11 ± 0.01 % vs \n0.05 ± 0.01 respectively). Smaller colonies also did not reveal \nany di fference between the woman without endometriosis and \nwomen with endometriosis (0.14 ± 0.02 % vs 0.06 ± 0.02 % \nrespectively) (Table 1).  \n \nTable 1: Colony Efficiency of Stromal Cells with and without Endometriosis \n \nSamples CE % P- Value Colonies CE % P- Value \nWith Endometriosis (n=15) 0.13 ± 0.01 \nP = 0.0007* \nLarge 0.05 ± 0.01 0.32** Small 0.06 ± 0.02 \nWithout Endometriosis (n=15) 0.25 ± 0.03 Large 0.11 ± 0.01 0.19** Small 0.14 ± 0.02 \n \n \n \nFig 1: Depicting that cells are positive for CD90 (Green in colour) which are loosely arranged and tightly arranged. But they are negative for CD34 \nbecause the cells are not stained with any colour. \n(a) Loosely arranged cells in colony showing positive expression for CD90-FITC and negative for CD34-PE with endometriosis (b) Tightly \narranged cells in colony showing highly positive expression for CD90-FITC and negative for CD34-PE without endometriosis \n \n \n     \n\nInternational Journal of Clinical Obstetrics and Gynaecology \n~ 202 ~ \n \n \n(2a) Small loosely-arranged colonies \n \n \n \n(2b) Large densely-packed colonies \n \n \n \n(2C) Characteristic Spindle-like morphology of the cultured cells are \npositive for CD90 \n \nFig 2: Adherent cells on day 4 \n \n6. Discussion \nThe biology of MSCs has been mainly studied due of its \ntherapeutic potential.  \nEndometrial stromal cells are located not only in the basalis, but \nalso in the functionalis, have the  ability to reconstruct \nendometrial tissue in vivo suggests their potential use for treating \ndisorders associated with inadequate endometrium. The \nidentification of specific markers for human endometrial MSC \nhas demonstrated their  perivascular location in the basalis and \nfunctionalis.  \n Our study provides the evaluation of Cells without Colony -\nForming in eutopic Endometrial progenitor Cells in infertile \nWomen with Endometriosis compared to healthy fertile women \nin Indian perspectiv e. The colony -forming ability of eutopic \nhuman endometrial samples with and without has been studied \nearlier without any significant differ ence in the total \nclonogeniety (Makarainen L 1988, Chan et al. 2011) [25, 5]. \nOur study shows the significant differe nce in the clonogenic \nprogenitor cells with p<0.05 in infertile women on comparision \nwith fertile women without endometriosis.  Hence there is no \nsignificant difference in large and small colonies in both the \ngroups. thus it can be assumed that the cells wi thout clonogenic \nstem cell cells play an important role in the abnormal function of \nendometrium during implantation. \nTherefore, we decided to study the function of CD90, one main \nimmunophenotypical marker of MSCs.CD90 has been identified \nas a candidate marker for MSCs. Endometriosis seems to have a \nnegative impact, on every part of the reproductive process subtly \nbut significantly. Infertility associated with endometriosis can be \neven more puzzling, as not every patient experiences the same \nsymptoms. \nThe hu man endometrium exhibits 0.15% of clonogenic \nepithelial and 1.3% stromal cell populations (Chan, R.W et al  \n2004, Schwab, K.E et al  2005) [6, 33 ]. Clonogenicity studies of \nhuman stromal stem/progenitor cells have been studied d by  in \nvitro in various differentiation assays \nCD90 is a potential marker for human endometrial stromal \nColony Forming Units that distinguishes basalis and functionalis \nstroma (Koumas L et al  2011). This study used CD90 in \ncombination with CD34 as a negative marker (multipotent) and \nshowed a trend to enrichment for CFU in the CD90 stromal cell \npopulation. Thus, the combination of CD90 with other markers \nmay increase its value for identifying and isolating purer \npopulations of endometrial stromal stem/progenitor cells. CD90 \nin MSCs repre sents a promising alternative strategy and an \nefficient approach to increase MSC differentiation efficiency in \nvitro; it may, therefore, be used in the future to improve MSC \ndifferentiation yields in cellular therapy.  \nIn conclusion, we have evaluated that  CD34, CD90 as \nhematopoietic markers of human endometrial stromal CFU. \nIncreasing number of studies has shown that MSCs from \ndifferent sources display significantly diverse properties and \ncharacteristics that may impact on their future therapeutic \napplications. The capacity of differentiation may vary according \nto the cell source (Schwab, K.E et al. 2005) [33]. CD90 may play \nan important role in maintaining the undifferentiated state of \nMSCs our findings indicate that a small population of \nendometriotic cel ls exhibits colony -forming activity, self -\nrenewal capacity, and multi potency. To determine any \ndifference between cells in endometriosis and those from \nendometrium, we compared the colony -forming activity of the \nendometrium and of the same patient, to avoid possible variation \ndue to individual difference in genetic background. Significantly \nmore clonogenic cells were detected from the endometrium  in \nour conclusion the study suggests that there is a significant \ndifference in the colony forming ability of th e eutopic \nendometrium cells in women with endometriosis in comparison \nwith the healthy fertile women in the Indian perspective.  \nThe eutopic endometrium from women with endometriosis \nshares certain alterations with ectopic lesions that are not \n\nInternational Journal of Clinical Obstetrics and Gynaecology \n~ 203 ~ \nobserved in the endometrium from healthy fertile women. It also \nprovides an understanding of not only the physiology of \nendometrium, but also the path physiology of endometrial \nendometriosis. The molecular and cellular mechanisms which \nare involved in the regulation o f progenitor cells in the eutopic \nendometrium in women with endometriosis leads to better \nunderstanding of Endometrial stem cell research is gaining \nmomentum and the knowledge generated may be translated into \nthe clinic within the next decade \n \n7. Authors Contribution  \nAll authors contributed equally to this work.  Dr. Roya Rozati \nalong with other authors discussed the methodology and results \nand also helped in preparing the manuscript at all stages.  \n \n8. References \n1. Akoum A, Metz CN, Al -Akoum M, Kats R . Macrophage \nmigration inhibitory factor expression in the intrauterine \nendometrium of women with endometriosis varies with \ndisease stage, infertility status and pelvic pain. Fertil Steril, \n2006; 85:1379-85 \n2. Allaire C. Endometriosis and infertility: A review. J Reprod \nMed. 2006; 51:164-168  \n3. Bondza PK, Maheux R, Akoum A. 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